Method of constructing a data architecture

ABSTRACT

A method of constructing a data architecture for use in identifying connections between perturbagens and genes associated with skin tone. The method includes providing a gene expression profile for a control cell, generating a gene expression profile for a cell exposed to a perturbagen, identifying genes differentially expressed in response to the perturbagen, creating an ordered list of identifiers, storing the ordered list as an instance on a computer readable medium, and constructing a data architecture of stored instances.

BACKGROUND OF THE INVENTION

Skin pigment irregularities are common across ethnic and racial groupsand are often considered cosmetically disfiguring. Disorders of pigmentproduction and distribution occur as a function of intensity andduration of UV radiation exposure, life style habits, chronological age,endocrine functioning and disease state and are found ubiquitously inolder populations. Hence there is a widespread demand for skin pigmentmodifying, skin lightening and skin tone enhancing products for thecosmetic market.

The color of normal human skin is due primarily to varying amounts anddistribution of melanin, hemoglobin, and carotenoids. Of these pigments,melanin is of primary significance to cosmetic skin treatment protocols.Melanin is produced by specialized cells in the skin called melanocytesthrough a complicated series of chemical and enzymatic reactions, mainlyinvolving the copper and manganese containing enzyme tyrosinase. Oncesynthesized, the melanin granules are packaged into melanosomes andtransferred via the cellular dendrites (extensions) of the melanocyte tothe surrounding keratinocytes, the most abundant cell type in theepidermis. The rate of melanin synthesis, and the subsequent transfer ofmelanin by melanocytes via their dendrites, appears to be influenced byultraviolet light exposure. Melanosomes transferred to the outer layerof the skin are responsible for the darkening of the skin, with thedegree of darkening being associated with skin type, sun exposure,and/or certain dermatological conditions.

Two types of melanin are present in human skin: (1) eumelanin, which isthe dark brown-black pigment found in most skin, hair, and eyes, andwhose production is stimulated by exposure to ultraviolet light, and (2)pheomelanin, which is a yellow-orange pigment found mainly in the skinof very fair-skinned people, particularly those with red hair. Theperceived color of skin is determined by the ratio of eumelanins topheomelanins, and to a smaller extent on blood within the dermis.

The pigmentation pathway has been elucidated in detail. Summarily,melanin forms through a series of oxidative reactions involving theamino acid tyrosine in the presence of the enzyme tyrosinase. Tyrosinaseconverts tyrosine to dihydroxyphenylalanine (DOPA) and then todopaquinone. Subsequently, dopaquinone is converted to dopachromethrough auto-oxidation, and finally to dihydroxyindole ordihydroxyindole-2-carboxylic acid (DHICA), which polymerize to formeumelanin. The latter reactions occur in the presence of dopachrometautomerase and DHICA oxidase. In the presence of sulfur-containingcysteine or glutathione, dopaquinone is converted to cysteinyl DOPA orglutathione DOPA; subsequently, pheomelanin is formed.

A variety of skin hyperpigmentation disorders are known and etiology isdiverse, overlapping in many cases, and often not fully understood. Forexample, melanosis or melasma is a condition characterized by thedevelopment of sharply demarcated blotchy, brown spots usually in asymmetric distribution over the cheeks, forehead, and sometimes on theupper lip and neck. This condition frequently occurs during pregnancy(melasma gravidarum or “mask of pregnancy”), and at menopause. Also,this condition is frequently found among those taking oralcontraceptives, and is occasionally found among nonpregnant women whoare not taking oral contraceptives, and sometimes among men. A patternof similar facial hyperpigmentation is associated with a chronic liverdisease called chloasma. A common condition associated with aging skinis the development of dark spots sometimes referred to as “age spots” or“liver spots.” Other forms of hyperpigmentation can be caused by UVirradiation, in particular UVB radiation which up-regulates theproduction of tyrosinase resulting in skin “tanning,” or may result froma genetic predisposition for the condition, or may come about inassociation with a skin inflammatory event or during the course of woundhealing.

Vitiligo is a form of hypopigmentation in which cutaneous melanocytesare either ablated or fail to produce sufficient pigment. Ideallytreatment would restore lost pigmentation in vitiligo-affected skin, butthis approach has met with little success via topical interventions andformulations. Although cosmetic camouflage with dihydroxyacetonesunless-tanning lotions provides some darkening of hypo-pigmented areas,it also tends to darken surrounding normal skin, substantiallymaintaining the undesirable contrast. Hence, a more favored cosmeticapproach is to reduce the normal pigmentation of the unaffected skin toreduce contrast and produce a tone evening effect.

Several proven targets for pigmentation control are known, but thesehave generally been derived from an understanding of the pigmentationprocess. Hydroquinone (parahydroxy-benzene), for example, is a widelyused skin lightening agent that is known to provide a satisfactorycosmetic result, however its use strictly for cosmetic purposes isdiscouraged due to its association with a variety of disorders,including diabetes, hypertension, ochronosis, periorbitary dyschromia,infectious dermatosis, contact eczema, extended dermatophytosis, andnecrotizing cellulites (see, e.g., Raynaud E. et al., Ann DermatolVenereol 128(6-7):720-724, 2001). Hydroquinone has also shown genotoxicand mutagenic activities (see, e.g., Jagetia G. C. et al, Toxicol Lett121(1):15-20, 2001). Due to concerns over toxicity and carcinogeniceffects, the United States limits treatment solutions to a 2% or lowerconcentration and the FDA has proposed a ban on all over-the-counterpreparations, while hydroquinone is currently banned in Europe as a skinlightening or depigmenting agent.

Kojic acid, Azelaic acid and certain-hydroxy acids such as glycolicacid, have demonstrated skin-lightening effects, but reports oflocalized irritation and inflammation are common. The prenylatedflavonol artocarpin has shown some efficacy for skin-lightening in thecontext of ultraviolet-induced skin pigmentation (Shimizu K. et al.,Planta Med 68(1):79-81, 2002).

Recently, a more detailed genomic and proteomic understanding ofmelanogenesis, the melanocyte, melanocyte-keratinocyte interaction, andthe melanocyte-fibroblast interaction has revealed potentially hundredsof proteins and other effectors involved in the pigmentation process andin the etiology of hyperpigmentation disorders, which may provideadditional targets. There is a need in the cosmetic arts both forgenerating potential skin lightening agents and for effective andefficient screening methods for identifying putative skin active agentswith efficacy and safety in the cosmetic treatment of hyperpigmentationand pigmentation disorders.

Traditionally scientists have focused on the development and provisionof safe and effective topical compositions formulated to lighten skinand such an approach has been useful for treating localized epidermalhyperpigmentation and for masking areas of skin hypopigmentation. Thereremains a need, however, for safe and effective agents capable ofdelivery through topical application to reduce the degree of skinpigmentation in both contexts.

Skin pigmentation and the broader cosmetic concept of skin tone, aretherefore highly complex conditions with multiple and overlappingetiologies, which manifest in part as a function of individualpredisposition, and which therefore pose a significant treatmentchallenge. There is a need in the art for methods of identifyingpotential skin pigment modifying agents, and in particularskin-lightening agents, and for evaluating the efficacy of putative skinactive agents using screening methods that are substantially independentof mechanism of action or etiology of the pigment condition. The presentinvestigators therefore undertook an investigation into the applicationof a relatively new technology known as “connectivity mapping” to thesearch for new skin-active agents with efficacy in the treatment ofhyperpigmentation disorders and related skin conditions.

Connectivity mapping is a well-known hypothesis generating and testingtool having successful application in the fields of operations research,telecommunications, and more recently in pharmaceutical drug discovery.The undertaking and completion of the Human Genome Project, and theparallel development of very high throughput high-density DNA microarraytechnologies enabling rapid and simultaneous quantization of cellularmRNA expression levels, resulted in the generation of an enormousgenetic database. At the same time, the search for new pharmaceuticalactives via in silico methods such as molecular modeling and dockingstudies stimulated the generation of vast libraries of potential smallmolecule actives. The amount of information linking disease to geneticprofile, genetic profile to drugs, and disease to drugs grewexponentially, and application of connectivity mapping as a hypothesistesting tool in the medicinal sciences ripened.

The general notion that functionality could be accurately determined forpreviously uncharacterized genes, and that potential targets of drugagents could be identified by mapping connections in a data base of geneexpression profiles for drug-treated cells, was spearheaded in 2000 withpublication of a seminal paper by T. R. Hughes et al. [“Functionaldiscovery via a compendium of expression profiles” Cell 102, 109-126(2000)], followed shortly thereafter with the launch of The ConnectivityMap (map Project by Justin Lamb and researchers at MIT (“ConnectivityMap: Gene Expression Signatures to Connect Small Molecules, Genes, andDisease”, Science, Vol 313, 2006.) In 2006, Lamb's group beganpublishing a detailed synopsis of the mechanics of C-map constructionand installments of the reference collection of gene expression profilesused to create the first generation C-map and the initiation of anon-going large scale community C-map project, which is available underthe “supporting materials” hyperlink at the sciencemag.org website.

The basic paradigm of predicting novel relationships between disease,disease phenotype, and drugs employed to modify the disease phenotype,by comparison to known relationships has been practiced for centuries asan intuitive science by medical clinicians. Modern connectivity mapping,with its rigorous mathematical underpinnings and aided by moderncomputational power, has resulted in confirmed medical successes withidentification of new agents for the treatment of various diseasesincluding cancer. Nonetheless, certain limiting presumptions challengeapplication of C-map with respect to diseases of polygenic origin orsyndromic conditions characterized by diverse and often apparentlyunrelated cellular phenotypic manifestations. According to Lamb, thechallenge to constructing a useful C-map is in the selection of inputreference data which permit generation of clinically salient and usefuloutput upon query. For the drug-related C-map of Lamb, strongassociations comprise the reference associations, and strongassociations are the desired output identified as hits.

Noting the benefit of high-throughput, high density profiling platformswhich permit automated amplification, labeling hybridization andscanning of 96 samples in parallel a day, Lamb nonetheless cautioned:“[e]ven this much firepower is insufficient to enable the analysis ofevery one of the estimated 200 different cell types exposed to everyknown perturbagen at every possible concentration for every possibleduration . . . compromises are therefore required” (page 54, column 3,last paragraph). Lamb, however, took the position that cell type did notultimately matter, and confined his C-map to data from a very smallnumber of established cell lines out of efficiency and standardizationconcerns. Theoretically this leads to heightened potential for in vitroto in vivo mismatch, and limits output information to the context of aparticular cell line. If one accepts the Lamb precept that cell linedoes not matter then this limitation may be benign.

However, agents suitable as pharmaceutical agents and agents suitable ascosmetic agents are categorically distinct, with the former definingagents selected for specificity and which are intended to havemeasurable effects on structure and function of the body, while thelatter are selected for effect on appearance and may not affectstructure and function of the body to a measurable degree. Cosmeticagents tend to be substantially non-specific with respect to effect oncellular phenotype, and administration to the body is generally limitedto application on or close to the body surface.

In constructing C-maps relating to pharmaceutical agents, Lamb stressesthat particular difficulty may be encountered if reference connectionsare extremely sensitive and at the same time difficult to detect (weak),and Lamb adopted compromises aimed at minimizing numerous, diffuseassociations. Since the regulatory scheme for drug products requireshigh degrees of specificity between a purported drug agent and diseasestate, and modulation of disease by impacting a single protein with aminimum of tangential associations is desired in development ofpharmaceutical actives, the Lamb C-map is well-suited for screening forpotential pharmaceutical agents despite the Lamb compromises.

The connectivity mapping protocols of Lamb would not be predicted,however, to have utility for hypothesis testing/generating in the fieldof cosmetics or for a primarily cosmetic disorder where symptoms may bediffuse, systemic and relatively mild. In complete contravention of thegoal of pharmaceutical active discovery, cosmetic formulators seekagents or compositions of agents capable of modulating multiple targetsand having effects across complex phenotypes and conditions. Further,the phenotypic impact of a cosmetic agent must be relatively low bydefinition, so that the agent avoids being subject to the regulatoryscheme for pharmaceutical actives. Nonetheless, the impact must beperceptible to the consumer and preferably empirically confirmable byscientific methods. Gene transcription/expression profiles for cosmeticconditions are generally diffuse, comprising many genes with low tomoderate fold differentials. Cosmetic agents, therefore, provide morediverse and less acute effects on cellular phenotype and generate thesort of associations expressly taught by Lamb as unsuitable forgenerating connectivity maps useful for confident hypothesis testing.

Successful identification of skin lightening agents has proven to bedifficult due to the multi-cellular, multi-factorial processes involvedin etiology of the hyperpigmentation condition itself. Conventional invitro studies of biological responses to potential skin-lighteningagents can be hindered by the complex or weakly detectable responsestypically induced and/or caused by the putative skin active or potentialskin active agents. Such weak responses arise, in part, due to the greatnumber of genes and gene products involved, and the fact thatskin-active and cosmetic agents may affect multiple genes in multipleways. Moreover, the degree of bioactivity of cosmetic agents may differfor each gene and be difficult to quantify.

The value of a connectivity map approach to discover functionalconnections among cosmetic phenotypes such as hyperpigmented skin, geneexpression perturbation, and cosmetic agent action is counter-indicatedby the progenors of the drug-based C-map. The relevant phenotypes arevery complex, the genetic perturbations are numerous and weak, andcosmetic agent action is likewise diffuse and by definition, relativelyweak. It is unclear whether statistically valid data may be generatedfrom cosmetic C-maps and it is further unclear whether a cell lineexists which may provide salient or detectable cosmetic data.

SUMMARY OF THE INVENTION

Accordingly, the present inventors have developed a C-map approach thatsurprisingly enables discovery of skin tone agents having efficacy fordisorders of skin pigmentation.

The present inventors discovered that useful connectivity maps could bedeveloped from cosmetic active—cellular phenotype—gene expression dataassociations in particular with respect to hyperpigmentation actives andcosmetic agents, despite the highly diffuse, systemic and low-leveleffects these sorts of actives generally engender. Although the Lambteam asserted that results should be substantially independent ofcell-type, the present inventors surprisingly discovered that selectionof cell line affects the utility of the C-map for hypothesis generatingand testing relating to skin pigmentation actives and treatment ofhyperpigmentation disorders. In particular, keratinocyte cells, ratherthan melanocyte or melanoma cells, exhibited a more robusttranscriptional profile when treated with skin-lightening agents, andthere was little to no thematic overlap between cell types treated withthe same benchmark skin active agent (shown in Example 8).

Accordingly, the present invention provides novel methods, systems andmodels useful for generating potential new skin-active agentsefficacious for the treatment of skin conditions such ashyperpigmentation. Through careful selection of cell type, and bygeneration of a reference collection of gene-expression profiles forknown skin-active agents and recognized skin disorders, the presentinventors were surprisingly able to create connectivity map architectureuseful for testing and generating hypotheses about skin-active agentsand hyperpigmentation skin disorders.

The present invention provides embodiments which broadly include methodsand systems for determining relationships between a skincondition/disorder of interest and one or more skin-active agents, oneor more genes associated with the skin disorder condition, andphysiological themes implicated by the skin condition and/or affected bya skin-active agent. The inventive methods may be used to identifyskin-active agents without detailed knowledge of the mechanisms ofbiological processes associated with a skin disorder or condition ofinterest, all of the genes associated with such a condition, or the celltypes associated with such a condition.

One aspect of the invention provides methods for constructing a dataarchitecture for use in identifying connections between perturbagens andgenes associated with skin tone, comprising: (a) providing a geneexpression profile for a control human cell, wherein the control cell isfrom a human cell line selected from the group consisting ofkeratinocyte, fibroblast, melanocyte and melanoma cell lines; (b)generating a gene expression profile for a human cell exposed to atleast one perturbagen, wherein the cell is selected from the same cellline as the control cell; (c) identifying genes differentially expressedin response to the at least one perturbagen by comparing the geneexpression profiles of (a) and (b); (d) creating an ordered listcomprising identifiers representing the differentially expressed genes,wherein the identifiers are ordered according to the differentialexpression of the genes; (e) storing the ordered list as an instance onat least one computer readable medium, wherein the instance is akeratinocyte, fibroblast melanocyte or melanoma instance according tothe selection in (a); and (f) constructing a data architecture of storedinstances by repeating (a) through (e), wherein the at least oneperturbagen of step (a) is different qualitatively or quantitatively foreach instance. In specific embodiments each instance is repeated twicein C-map testing. Example 4 illustrates generating a benchmark skin toneagent signature using fibroblasts to create signatures and usingkeratinocytes to create signatures.

The data architecture may be mined to identify relationships betweenperturbagens, genotypes and phenotypes and may also be used as an insilico tool for generating new actives with potential efficacy fortreatment of a cosmetic condition. The data architecture may beimplemented for this purpose through the use of a query, which is aninput to the C-map wherein the output is based on connectivity scores tothe query. In one embodiment, a method for implementing the dataarchitecture to identify at least one putative skin active agent havingpotential efficacy in treating a skin pigmentation condition isprovided. The method comprises querying the data architecture with apigmentation-relevant gene expression signature, wherein queryingcomprises comparing the pigmentation-relevant gene expression signatureto each stored cell instance, and further wherein thepigmentation-relevant expression signature represents genesdifferentially expressed in cells derived from skin affected with a skinhyperpigmentation condition or genes differentially expressed in cellstreated with at least one benchmark skin active agent having knownefficacy in treating a skin hyperpigmentation condition. Cell instancesare derived from a keratinocyte, fibroblast, melanocyte or melanoma cellline and the pigmentation-relevant gene expression signature is derivedfrom a corresponding cell line. Example 1 illustrates development of ahyperpigmentation condition expression signature.

Other embodiments are direct to methods for generating ahyperpigmentation condition gene expression signature for use inidentifying connections between perturbagens and genes associated with askin pigmentation condition. Methods comprise: (a) providing a geneexpression profile for a reference sample of human skin cells notaffected with a pigmentation condition; (b) generating a gene expressionprofile for at least one sample of human skin cells from a subjectexhibiting the hyperpigmentation condition, (c) comparing the expressionprofiles of (a) and (b) to determine a gene expression signaturecomprising a set of genes differentially expressed in (a) and (b); (d)assigning an identifier to each gene constituting the gene expressionsignature and ordering the identifiers according to the direction ofdifferential expression to create one or more gene expression signaturelists; and (e) storing the one or more gene expression signature listson at least one computer readable medium.

Another embodiment provides methods for generating a benchmark skinpigmentation-modifying gene expression signature for use in identifyingconnections between perturbagens and genes associated with a skinpigmentation condition, the method comprising: (a) generating a geneexpression profile for a human skin cell sample treated with at leastone benchmark skin pigmentation modifying agent, wherein the benchmarkskin pigmentation modifying agent is suspended in a vehicle composition,(b) generating a gene expression profile for a human skin cell sampletreated with the vehicle composition; (c) comparing the expressionprofiles of (a) and (b) to determine a gene expression signaturecomprising a set of genes differentially expressed in (a) and (b); (d)assigning an identifier to each gene constituting the gene expressionsignature and ordering the identifiers according to the direction ofdifferential expression to create one or more gene expression signaturelists; and (e) storing the one or more gene expression signature listson at least one computer readable medium. Gene expression signatures andimmobilized arrays of probes corresponding to the genes constituting theinventive signatures are also provided.

In some aspects a single benchmark skin active agent may be used togenerate a benchmark signature (see Example 2) and in other aspects acomposite signature may be generated by treating a cell sample with morethan one agent (see Examples 3, 6, and 7). A composite signature can beadded in two ways: cells can be treated with each agent separately, thesignature can be generated by comparing regulated genes from all agents(together), looking for genes regulated in the same direction by allagents; secondarily, agents can be mixed together prior to treatment ofcells. In another embodiment, a composite benchmark signature may begenerated for a skin-lightening agent (Example 2), and another generatedfor a skin darkening agent. The signature for the skin-lightening agentmay be further tweaked by eliminating any gene from the signature thatalso appears in the signature of the skin-darkening agent, regulated inthe same direction, or vice versa. The inventors discovered that suchcomposite signatures are particularly useful for mining C-map for agentscapable of modifying skin pigment in the desired direction.

Systems for identifying connections between perturbagens and genesassociated with a skin hyperpigmentation condition are also provided.The systems comprise: (a) at least one computer readable medium havingstored thereon a plurality of instances, and a skinhyperpigmentation-relevant gene expression signature, wherein theinstances and the gene expression signature are derived from one of ahuman keratinocyte cell, a human fibroblast cell, a human melanocytecell, or a human melanoma cell, wherein each instance comprises aninstance list of rank-ordered identifiers of differentially expressedgenes, and wherein the hyperpigmentation-relevant gene expressionsignature comprises one or more gene expression signature lists ofidentifiers representing differentially expressed genes associated witha hyperpigmentation condition or differentially expressed genesassociated with a benchmark skin-lightening agent; (b) a programmablecomputer comprising computer-readable instructions that cause theprogrammable computer to execute one or more of the following: (i)accessing the plurality of instances and a hyperpigmentation-relevantgene expression signature stored on the computer readable medium; (ii)comparing the hyperpigmentation-relevant gene expression signature tothe plurality of the instances, wherein the comparison comprisescomparing each identifier in the gene expression signature list with theposition of the same identifier in the instance list for each of theplurality of instances; and (iii) assigning a connectivity score to eachof the plurality of instances.

A computer readable medium aspect is also disclosed wherein a dataarchitecture comprising a first digital file is stored in a spreadsheetfile format, a word processing file format, or a database file formatsuitable to be read by a respective spreadsheet, word processing, ordatabase computer program, the first digital file comprising dataarranged to provide one or more gene expression signature listscomprising a plurality of identifiers when read by the respectivespreadsheet, word processing, or database computer program; and whereineach identifier is selected from the group consisting of a microarrayprobe set ID, a human gene name, a human gene symbol, and combinationsthereof representing a gene set forth in any of Tables B through Pwherein each of the one or more gene expression signature listscomprises between about 10 and about 400 identifiers. Instructions forreading the digital file may be included.

These and additional objects, embodiments, and aspects of the inventionwill become apparent by reference to the Figures and DetailedDescription below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 sets forth pigmentation control targets and some benchmark skinactive agents as depicted in Table A.

FIG. 2 sets forth identifiers for the genes constituting a geneexpression signature for hyperpigmented skin. Table B sets forthidentifiers for the 200 most significantly up-regulated genes and TableC sets forth identifiers for the 200 most significantly down-regulatedgenes.

FIG. 3 is a schematic illustration of a computer system suitable for usewith the present invention;

FIG. 4 is a schematic illustration of an instance associated with acomputer readable medium of the computer system of FIG. 3;

FIG. 5 is a schematic illustration of a programmable computer suitablefor use with the present invention;

FIG. 6 is a schematic illustration of an exemplary system for generatingan instance;

FIG. 7 is a schematic illustration of a comparison between a geneexpression signature and an instance, wherein there is a positivecorrelation between the lists;

FIG. 8 is a schematic illustration of a comparison between a geneexpression signature and an instance, wherein there is a negativecorrelation between the lists; and

FIG. 9 is a schematic illustration of a comparison between a geneexpression signature and an instance, wherein there is a neutralcorrelation between the lists.

FIG. 10 sets forth identifiers as shown in Table D, Hexamidine BenchmarkSignature; 100 up-regulated.

FIG. 11 sets forth identifiers as shown in Table E, Hexamidine BenchmarkSignature; 100 top down-regulated.

FIG. 12 sets forth identifiers as shown in Table F, NAG BenchmarkSignature; 39 significantly up-regulated.

FIG. 13 sets forth identifiers as shown in Table G, NAG BenchmarkSignature, most significantly down-regulated.

FIG. 14 sets forth identifiers as shown in Table H, NiacinamideBenchmark Signature, 100 top up-regulated.

FIG. 15 sets forth identifiers as shown in Table I, NiacinamideBenchmark Signature, 100 top down-regulated.

FIG. 16 sets forth identifiers as shown in Table J, Sepiwhite BenchmarkSignature; 100 top up-regulated.

FIG. 17 sets forth identifiers as shown in Table K, Sepiwhite BenchmarkSignature; 100 top down-regulated.

FIG. 18 sets forth identifiers as shown in Table L, Composite “SkinTone” Benchmark Signature; approximately 40 up-regulated+58down-regulated.

FIG. 19 sets forth identifiers as shown in Table M, RA benchmarksignature in tKC cell—200 upregulated.

FIG. 20 sets forth identifiers as shown in Table N, RA benchmarksignature in tKC, 200 down-regulated.

FIG. 21 sets forth identifiers as shown in Table O, RA BenchmarkSignature in BJ fibroblasts, 200 up-regulated.

FIG. 22 sets forth identifiers as shown in Table P, RA BenchmarkSignature in BJ fibroblast, 200 down-regulated.

FIG. 23 sets in Table Q, Average C-map scores for some representativepotential skin lightening agents with the Retinoic Acid KeratinocyteRA_200 Signature

FIG. 24 Shows Table R, showing a comparison of the predictiveness ofdifferent C-map signatures for predicting the activity of compounds inthe mouse B16 melanoma cell melanogenesis assay

FIG. 25, Shows Table S, with data outlining the responsiveness of tertKeratinocytes, melanocytes, and melanoma cells to skin tone benchmarks.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with occasional reference tothe specific embodiments of the invention. This invention may, however,be embodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete, and to fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention. Asused in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used interchangeably herein, the terms “connectivity map” and “C-map”refer broadly to devices, systems, articles of manufacture, andmethodologies for identifying relationships between cellular phenotypesor cosmetic conditions, gene expression, and perturbagens, such ascosmetic actives.

As used herein, the term “cosmetic agent” means any substance, as wellas any component thereof, which may be rubbed, poured, sprinkled,sprayed, introduced into, or otherwise applied to a mammalian body orany part thereof. Cosmetic agents may include substances that areGenerally Recognized as Safe (GRAS) by the US Food and DrugAdministration, food additives, and materials used in non-cosmeticconsumer products including over-the-counter medications. In someembodiments, cosmetic agents may be incorporated in a cosmeticcomposition comprising a dermatologically acceptable carrier suitablefor topical application to skin. A cosmetic agent includes, but is notlimited to, (i) chemicals, compounds, small or large molecules,extracts, formulations, or combinations thereof that are known to induceor cause at least one effect (positive or negative) on skin tissue; (ii)chemicals, compounds, small molecules, extracts, formulations, orcombinations thereof that are known to induce or cause at least oneeffect (positive or negative) on skin tissue and are discovered, usingthe provided methods and systems, to induce or cause at least onepreviously unknown effect (positive or negative) on the skin tissue; and(iii) chemicals, compounds, small molecules, extracts, formulations, orcombinations thereof that are not known have an effect on skin tissueand are discovered, using the provided methods and systems, to induce orcause an effect on skin tissue.

Some examples of cosmetic agents or cosmetically actionable materialscan be found in: the PubChem database associated with the NationalInstitutes of Health, USA; the Ingredient Database of the Personal CareProducts Council; and the 2010 International Cosmetic IngredientDictionary and Handbook, 13^(th) Edition, published by The Personal CareProducts Council; the EU Cosmetic Ingredients and Substances list; theJapan Cosmetic Ingredients List; the Personal Care Products Council, theSkinDeep database; the FDA Approved Excipients List; the FDA OTC List;the Japan Quasi Drug List; the US FDA Everything Added to Food database;EU Food Additive list; Japan Existing Food Additives, Flavor GRAS list;US FDA Select Committee on GRAS Substances; US Household ProductsDatabase; the Global New Products Database (GNPD) Personal Care, HealthCare, Food/Drink/Pet and Household database; and from suppliers ofcosmetic ingredients and botanicals.

Other non-limiting examples of cosmetic agents include botanicals (whichmay be derived from one or more of a root, stem bark, leaf, seed orfruit of a plant). Some botanicals may be extracted from a plant biomass(e.g., root, stem, bark, leaf, etc.) using one more solvents. Botanicalsmay comprise a complex mixture of compounds and lack a distinct activeingredient. Another category of cosmetic agents are vitamin compoundsand derivatives and combinations thereof, such as a vitamin B3 compound,a vitamin B5 compound, a vitamin B6 compound, a vitamin B9 compound, avitamin A compound, a vitamin C compound, a vitamin E compound, andderivatives and combinations thereof (e.g., retinol, retinyl esters,niacinamide, folic acid, panethenol, ascorbic acid, tocopherol, andtocopherol acetate). Other non-limiting examples of cosmetic agentsinclude sugar amines, phytosterols, hexamidine, hydroxy acids,ceramides, amino acids, and polyols.

Non-limiting examples of agents herein utilized are described in detailbelow, such as for vitamin B3 compounds, N-acyl amino acid compounds,and retinoid compounds. In some embodiments, the vitamin B compound is aB3 compound having the formula:

wherein R is —CONH₂ (i.e., niacinamide), —COOH (i.e., nicotinic acid) or—CH2OH (i.e., nicotinyl alcohol); derivatives thereof; and salts of anyof the foregoing. Exemplary derivatives include nicotinic acid esters,including non-vasodilating esters of nicotinic acid (e.g., tocopherylnicotinate, myristyl nicotinate). Examples of suitable vitamin B₃compounds are well known in the art and are commercially available froma number of sources (e.g., the Sigma Chemical Company, ICN Biomedicals,Inc., and Aldrich Chemical Company).

Some embodiments of the compositions of the present invention comprise asafe and effective amount of one or more N-acyl amino acid compounds.The amino acid can be one of any of the amino acids known in the art.The N-acyl amino acid compounds of the present invention correspond tothe formula:

wherein R can be a hydrogen, alkyl (substituted or unsubstituted,branched or straight chain), or a combination of alkyl and aromaticgroups. A list of possible side chains of amino acids known in the artare described in Stryer, Biochemistry, 1981, published by W.H. Freemanand Company. R¹ can be C₁ to C₃₀, saturated or unsaturated, straight orbranched, substituted or unsubstituted alkyls; substituted orunsubstituted aromatic groups; or mixtures thereof.

Preferably, the N-acyl amino acid compound is selected from the groupconsisting of N-acyl Phenylalanine, N-acyl Tyrosine, their isomers,their salts, and derivatives thereof. The amino acid can be the D or Lisomer or a mixture thereof. N-acyl Phenylalanine corresponds to thefollowing formula:

wherein R¹ can be C₁ to C₃₀, saturated or unsaturated, straight orbranched, substituted or unsubstituted alkyls; substituted orunsubstituted aromatic groups; or mixtures thereof.

N-acyl Tyrosine corresponds to the following formula:

wherein R¹ can be C₁ to C₃₀, saturated or unsaturated, straight orbranched, substituted or unsubstituted alkyls; substituted orunsubstituted aromatic groups; or mixtures thereof.

A particularly useful compound in the present invention isN-undecylenoyl-L-phenylalanine. This agent belongs to the broad class ofN-acyl Phenylalanine derivatives, with its acyl group being a C11mono-unsaturated fatty acid moiety and the amino acid being the L-isomerof phenylalanine. N-undecylenoyl-L-phenylalanine corresponds to thefollowing formula:

As used herein, N-undecylenoyl-L-phenylalanine is commercially availableunder the tradename Sepiwhite® from SEPPIC, France.

Some embodiments of the present invention include retinoid compounds. Asused herein, “Retinoid Compounds” include all natural and/or syntheticanalogs of Vitamin A or retinol-like compounds which possess thebiological activity of Vitamin A in the skin as well as the geometricisomers and stereoisomers of these compounds. The Retinoid Compoundincludes, but is not limited to, retinol, retinol esters (e.g., C₂-C₂₂alkyl esters of retinol, including retinyl palmitate, retinyl acetate,retinyl proprionate), retinal, and/or retinoic acid (including all-transretinoic acid and/or 13-cis-retinoic acid). In some embodiments, theRetinoid Compound is retinoic acid. These compounds are well known inthe art and are commercially available from a number of sources, e.g.,Sigma Chemical Company (St. Louis, Mo.), and Boerhinger Mannheim(Indianapolis, Ind.). Other Retinoid Compounds which may be usefulherein are described in U.S. Pat. No. 4,677,120, issued Jun. 30, 1987 toParish et al.; U.S. Pat. No. 4,885,311, issued Dec. 5, 1989 to Parish etal.; U.S. Pat. No. 5,049,584, issued Sep. 17, 1991 to Purcell et al.;U.S. Pat. No. 5,124,356, issued Jun. 23, 1992 to Purcell et al.; andReissue 34,075, issued Sep. 22, 1992 to Purcell et al.

As used herein, the term “putative skin active agent” means a cosmeticagent as herein defined that has shown promise through preliminaryscreens as effecting a specific change in skin biology related topigmentation but that has not yet been tested for effectiveness throughthe methods herein described for constructing a data architecture foruse in identifying connections between perturbagens and genes associatedwith skin tone, comprising

As used herein, the term “skin-active agent” is a subset of cosmeticagents as defined herein and includes generally any substance, as wellas any component thereof, intended to be applied to the skin for thepurpose of effectuating a treatment of an undesirable skin condition ordisorder, or for achieving a desirable skin status. Examples relating toskin tone include skin pigmentation disorders, including disorders ofhyperpigmentation, such as ephelides (freckles), lentigines includingage spots (solar lentigos), post-inflammatory hyperpigmentation, Café aulait macules, Addisons disease and other systemic disease effects,hemochromatosis, melasma (mask of pregnancy and other hormonal relatedpigment disorders) and acanthosis nigricans, as well as phototoxia andmedicinal-induced alternations in pigmentation. Examples of disorders ofhypopigmentation include Vitiligo and skin trauma-related ablation ofmelanocytes in circumscribed areas. In some case a cosmetic consumermerely desires a change in pigmentation status of skin as determined bysome cultural standard, such as skin lightening among some dark skinnedpeople and skin darkening or tanning among some light skinned people.

Although the term “skin tone” is most often thought of with respect toskin pigmentation and evenness of coloration, “skin tone” may alsoinclude other characteristics of skin that contribute to a consumerperception of overall tone. For example, pore size and distribution, andskin texture are also generally considered attributes of overall skintone.

Categorical examples of skin-active agents include skin pigmentmodifying agents, steroidal anti-inflammatory agents, non-steroidalanti-inflammatory agents, pediculocides, sensates, enzymes, vitamins,hair growth actives, sunscreens, and combinations thereof. Cosmeticcompositions according to the instant invention may contain skin-activeagents.

Many processes and proteins are known to be involved in the pigmentaryprocess; there is a wide array of targets against which to screen forpigmentation control agents. Among the many targets are inhibitors ofmelanocyte stimulation (e.g., antioxidants, anti-inflammatory agents),cell receptor antagonists (e.g., alpha-MSH antagonists), inhibitors ofmelanin synthesis enzymes (e.g., tyrosinase, TRP-1, TRP-2), inhibitorsof melanosome transport within the melanocyte and transfer to thekeratinocyte (e.g., PAR-2 antagonists), and activators of melanindegradation within the keratinocyte.

Skin active agents which modify skin pigmentation are known in the art.Certain substances may only be considered cosmetic in severely reducedconcentrations since there are side effects which suggest undesirablesystemic activity beyond the cosmetic concern being addressed. Theseinclude hydroquinone, trans-retinoic acid, and corticosteroids.Generally, a classic target for cosmetic formulation is inhibition oftyrosinase, the first enzyme in the conversion of tyrosine to melanin. Awide array of compounds, such as kojic acid, arbutin, ascorbic acid,ellagic acid, sulfhydryl compounds, and resorcinols, are effectivetyrosinase inhibitors, as is a more recently discussed deoxy-arbutin.However, since several of these materials also have other effects, it isdifficult to directly connect a specific mechanism to the observedeffect on pigmentation. Table A provides a short list of the many knowntargets and a few agents effective against them.

Generally, a “benchmark skin active agent” refers to any chemical,compound, environmental factor, small or large molecule, extract,formulation, or combinations thereof that is known to induce or cause asuperior effect (positive or negative) on skin tissue. In accordancewith the present invention, agents having known efficacy in eitherskin-lightening or skin darkening contexts are also herein referred toas “Benchmark Agents.”

Non-limiting examples of benchmark skin active agents are set forth inTable A, along with the corresponding theorized pigmentation controltarget.

Newer benchmark skin active agents include niacinamide and glucosamine(in particular, its derivative N-acetyl glucosamine [NAG]), which haverecently been shown to be effective in reducing melanin production inculture. In vitro, glucosamine reduces production of melanin byinhibiting activation of tyrosinase, while niacinamide inhibitsmelanosome transfer from melanocytes to keratinocytes. Cosmeticmoisturizer formulations containing niacinamide alone are effective inreducing the appearance of hyperpigmented spots in vivo and the additionof NAG to the formula yields greater effectiveness. Another benchmarkpigmentation control agent is N-undecylenoyl-L-phenylalanine, which hasbeen reported to inhibit biding of alpha-MSH to the melanocyte in vitroand is effective as a component of cosmetic moisturizer formulations inclinical testing.

The terms “gene expression signature,” and “gene-expression signature”refer to a rationally derived list, or plurality of lists, of genesrepresentative of a skin tissue condition or a skin agent. In specificcontexts, the skin agent may be a benchmark skin active agent or apotential skin agent. Thus, the gene expression signature may serve as aproxy for a phenotype of interest for skin tissue. A gene expressionsignature may comprise genes whose expression, relative to a normal orcontrol state, is increased (up-regulated), whose expression isdecreased (down-regulated), and combinations thereof. Generally, a geneexpression signature for a modified cellular phenotype may be describedas a set of genes differentially expressed in the modified cellularphenotype compared to the cellular phenotype. A gene expressionsignature can be derived from various sources of data, including but notlimited to, from in vitro testing, in vivo testing and combinationsthereof. In some embodiments, a gene expression signature may comprise afirst list representative of a plurality of up-regulated genes of thecondition of interest and a second list representative of a plurality ofdown-regulated genes of the condition of interest.

As used herein, the term “query” refers to data that is used as an inputto a Connectivity Map and against which a plurality of instances arecompared. A query may include a gene expression signature associatedwith a skin condition such as age spots, or may include a geneexpression signature derived from a physiological process associatedwith a skin condition. A C-map may be queried with perturbagens, geneexpression signatures, skin disorders, thematic signatures, or any datafeature or combination of data features or associations that comprisethe data architecture.

The term “instance,” as used herein, refers to data from a geneexpression profiling experiment in which skin cells are dosed with aperturbagen. In some embodiments, the data comprises a list ofidentifiers representing the genes that are part of the gene expressionprofiling experiment. The identifiers may include gene names, genesymbols; microarray probe set IDs, or any other identifier. In someembodiments, an instance may comprise data from a microarray experimentand comprises a list of probe set IDs of the microarray ordered by theirextent of differential expression relative to a control. The data mayalso comprise metadata, including but not limited to data relating toone or more of the perturbagen, the gene expression profiling testconditions, the skin cells, and the microarray.

The term “perturbagen,” as used herein, means anything used as achallenge in a gene expression profiling experiment to generate geneexpression data for use in the present invention. In some embodiments,the perturbagen is applied to human cells and the gene expression dataderived from the gene expression profiling experiment may be stored asan instance in a data architecture. Human cells in accordance with theinvention may be keratinocyte, melanocyte, fibroblast, or melanomacells. Any substance, chemical, compound, active, natural product,extract, drug [e.g. Sigma-Aldrich LOPAC (Library of PharmacologicallyActive Compounds) collection], small molecule, and combinations thereofused as to generate gene expression data can be a perturbagen. Aperturbagen can also be any other stimulus used to generate differentialgene expression data. For example, a perturbagen may also be UVradiation, heat, osmotic stress, pH, a microbe, a virus, and smallinterfering RNA. A perturbagen may be, but is not required to be, anycosmetic agent.

The term “dermatologically acceptable,” as used herein, means that thecompositions or components described are suitable for use in contactwith human skin tissue.

As used herein, the term “computer readable medium” refers to anyelectronic storage medium and includes but is not limited to anyvolatile, nonvolatile, removable, and non-removable media implemented inany method or technology for storage of information such as computerreadable instructions, data and data structures, digital files, softwareprograms and applications, or other digital information. Computerreadable media includes, but are not limited to, application-specificintegrated circuit (ASIC), a compact disk (CD), a digital versatile disk(DVD), a random access memory (RAM), a synchronous RAM (SRAM), a dynamicRAM (DRAM), a synchronous DRAM (SDRAM), double data rate SDRAM (DDRSDRAM), a direct RAM bus RAM (DRRAM), a read only memory (ROM), aprogrammable read only memory (PROM), an electronically erasableprogrammable read only memory (EEPROM), a disk, a carrier wave, and amemory stick. Examples of volatile memory include, but are not limitedto, random access memory (RAM), synchronous RAM (SRAM), dynamic RAM(DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM),and direct RAM bus RAM (DRRAM). Examples of non-volatile memory include,but are not limited to, read only memory (ROM), programmable read onlymemory (PROM), erasable programmable read only memory (EPROM), andelectrically erasable programmable read only memory (EEPROM). A memorycan store processes and/or data. Still other computer readable mediainclude any suitable disk media, including but not limited to, magneticdisk drives, floppy disk drives, tape drives, Zip drives, flash memorycards, memory sticks, compact disk ROM (CD-ROM), CD recordable drive(CD-R drive), CD rewriteable drive (CD-RW drive), and digital versatileROM drive (DVD ROM).

As used herein, the terms “software” and “software application” refer toone or more computer readable and/or executable instructions that causea computing device or other electronic device to perform functions,actions, and/or behave in a desired manner. The instructions may beembodied in one or more various forms like routines, algorithms,modules, libraries, methods, and/or programs. Software may beimplemented in a variety of executable and/or loadable forms and can belocated in one computer component and/or distributed between two or morecommunicating, co-operating, and/or parallel processing computercomponents and thus can be loaded and/or executed in serial, parallel,and other manners. Software can be stored on one or more computerreadable medium and may implement, in whole or part, the methods andfunctionalities of the present invention.

As used herein, the term “hyperpigmentation gene expression signature”refers to a gene expression signature derived from gene expressionprofiling of a hyperpigmentation condition.

As used herein, the term “connectivity score” refers to a derived valuerepresenting the degree to which an instance correlates to a query.

As used herein, the term “data architecture” refers generally to one ormore digital data structures comprising an organized collection of data.In some embodiments, the digital data structures can be stored as adigital file (e.g., a spreadsheet file, a text file, a word processingfile, a database file, etc.) on a computer readable medium. In someembodiments, the data architecture is provided in the form of a databasethat may be managed by a database management system (DBMS) that is beused to access, organize, and select data (e.g., instances and geneexpression signatures) stored in a database.

As used herein, the terms “gene expression profiling” and “geneexpression profiling experiment” refer to the measurement of theexpression of multiple genes in a biological sample using any suitableprofiling technology. For example, the mRNA expression of thousands ofgenes may be determined using microarray techniques. Other emergingtechnologies that may be used include RNA-Seq or whole transcriptomesequencing using NextGen sequencing techniques.

As used herein, the term “microarray” refers broadly to any orderedarray of nucleic acids, oligonucleotides, proteins, small molecules,large molecules, and/or combinations thereof on a substrate that enablesgene expression profiling of a biological sample. Non-limiting examplesof microarrays are available from Affymetrix, Inc.; AgilentTechnologies, Inc.; Illumina, Inc.; GE Healthcare, Inc.; AppliedBiosystems, Inc.; Beckman Coulter, Inc.; etc.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about”.Additionally, the disclosure of any ranges in the specification andclaims are to be understood as including the range itself and alsoanything subsumed therein, as well as endpoints. All numeric ranges areinclusive of narrower ranges; delineated upper and lower range limitsare interchangeable to create further ranges not explicitly delineated.Unless otherwise indicated, the numerical properties set forth in thespecification and claims are approximations that may vary depending onthe desired properties sought to be obtained in embodiments of thepresent invention. Notwithstanding that numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical values, however, inherently containcertain errors necessarily resulting from error found in theirrespective measurements.

In accordance with one aspect of the present invention, provided aredevices, systems and methods for implementing a connectivity maputilizing one or more query signatures associated with a pigmentation orpigmentation-related condition. The query signatures may be derived invariety of ways. In some embodiments, the query signatures may be geneexpression signatures derived from gene expression profiling of fullthickness skin biopsies of skin exhibiting a skin condition of interestcompared to a control. The gene expression profiling can be carried outusing any suitable technology, including but not limited to microarrayanalysis or NextGen sequencing. An example of a gene expressionsignature includes a hyperpigmentation gene expression signature, anexample of which is described more fully hereafter. A query signaturemay be derived from transcriptional profiling of a keratinocyte,fibroblast, melanocyte, or melanoma cell line exposed to benchmarkskin-active agents such as skin-lightening agents. In other embodiments,the query signature may be a benchmark gene expression signature whereina skin-lightening benchmark signature is further refined by comparing itto a skin-darkening benchmark signature and genes having similardirectional regulation are eliminated. In further embodiments a cell istreated with more than one benchmark skin active agent to derive abenchmark composite signature, and in specific embodiments, the cell istreated with a plurality of benchmark skin active agents wherein theselected agents include those acting from different mechanisms known tounderpin skin pigmentation. In other specific embodiments a generalbenchmark skin tone signature may be generated by treating a cell with aplurality of benchmark skin active agents including agents comprisingbenchmark skin active agents for skin pigmentation, skin pore size anddistribution, and/or skin texture. These query signatures may be usedsingularly or in combination. In specific embodiments a compositesignature has been shown to provide advantages in predicting genechanges for chemicals affecting tone versus signatures from singlechemicals.

In accordance with another aspect of the present invention, provided aredevices, systems, and methods for implementing a connectivity maputilizing one or more instances derived from a perturbagen, such as acosmetic agent, exposed to an epidermal or dermal cell line, includingfor example keratinocyte, fibroblast, melanocyte and melanoma cells.Instances from more complex cell culture systems may also be used, suchas skin organotypic cultures containing the targeted cell or ex vivohuman skin. Instances from a plurality of cell lines may be used withthe present invention.

In accordance with yet another aspect of the present invention, providedare devices, systems and methods for identification of relationshipsbetween a skin condition, e.g. skin hyperpigmentation condition querysignature and a plurality of instances, where the query signature may bea gene expression signature or a physiological theme expressionsignature. For example, it may be possible to ascertain perturbagensthat give rise to a statistically significant activity on astatistically significant number of genes associated with a skincondition of interest, leading to the identification of new cosmeticagents for treating the skin condition or new uses of known cosmeticagents.

I. Systems and Devices

Referring to FIGS. 3, 5 and 6, some examples of systems and devices inaccordance with the present invention for use in identifyingrelationships between perturbagens, skin pigmentation conditions, andgenes associated with the skin pigmentation condition will now bedescribed. System 10 comprises one or more of computing devices 12, 14,a computer readable medium 16 associated with the computing device 12,and communication network 18.

The computer readable medium 16, which may be provided as a hard diskdrive, comprises a digital file 20, such as a database file, comprisinga plurality of instances 22, 24, and 26 stored in a data structureassociated with the digital file 20. The plurality of instances may bestored in relational tables and indexes or in other types of computerreadable media. The instances 22, 24, and 26 may also be distributedacross a plurality of digital files, a single digital file 20 beingdescribed herein however for simplicity.

The digital file 20 can be provided in wide variety of formats,including but not limited to a word processing file format (e.g.,Microsoft Word), a spreadsheet file format (e.g., Microsoft Excel), anda database file format. Some common examples of suitable file formatsinclude, but are not limited to, those associated with file extensionssuch as *.xls, *.xld, *.xlk, *.xll, *.xlt, *.xlxs, *.dif, *.db, *.dbf,*.accdb, *.mdb, *.mdf, *.cdb, *.fdb, *.csv, *sql, *.xml, *.doc, *.txt,*.rtf, *.log, *.docx, *.ans, *.pages, *.wps, etc.

Referring to FIG. 4, in some embodiments the instance 22 may comprise anordered listing of microarray probe set IDs, wherein the value of N isequal to the total number of probes on the microarray used in analysis.Common microarrays include Affymetrix GeneChips and Illumina BeadChips,both of which comprise probe sets and custom probe sets. To generate thereference gene profiles according to the invention, preferred chips arethose designed for profiling the human genome. Examples of Affymetrixchips with utility in the instant invention include model Human GenomeHG-U133 Plus 2.0 and HG-U219. A specific Affymetrix chip employed by theinstant investigators is HG-U133A2.0, however it will be understood by aperson or ordinary skill in the art that any chip or microarray,regardless of proprietary origin, is suitable so long as the probe setsof the chips used to construct a data architecture according to theinvention are substantially similar.

Instances derived from microarray analyses utilizing AffymetrixGeneChips may comprise an ordered listing of gene probe set IDs wherethe list comprises 22,000+ IDs. The ordered listing may be stored in adata structure of the digital file 20 and the data arranged so that,when the digital file is read by the software application 28, aplurality of character strings are reproduced representing the orderedlisting of probe set IDs. While it is preferred that each instancecomprise a full list of the probe set IDs, it is contemplated that oneor more of the instances may comprise less than all of the probe set IDsof a microarray. It is also contemplated that the instances may includeother data in addition to or in place of the ordered listing of probeset IDs. For example, an ordered listing of equivalent gene names and/orgene symbols may be substituted for the ordered listing of probe setIDs. Additional data may be stored with an instance and/or the digitalfile 20. In some embodiments, the additional data is referred to asmetadata and can include one or more of cell line identification, batchnumber, exposure duration, and other empirical data, as well as anyother descriptive material associated with an instance ID. The orderedlist may also comprise a numeric value associated with each identifierthat represents the ranked position of that identifier in the orderedlist.

Referring again to FIGS. 3, 4 and 5, the computer readable medium 16 mayalso have a second digital file 30 stored thereon. The second digitalfile 30 comprises one or more lists 32 of microarray probe set IDsassociated with one or more pigmentation-relevant gene expressionsignatures. The listing 32 of microarray probe set IDs typicallycomprises a much smaller list of probe set IDs than the instances of thefirst digital file 20. In some embodiments, the list comprises between 2and 1000 probe set IDs. In other embodiments the list comprises greaterthan 10, 50, 100, 200, or 300 and/or less than about 800, 600, or about400 probe set IDs. The listing 32 of probe set IDs of the second digitalfile 30 comprises a list of probe set IDs representing up, and/ordown-regulated genes selected to represent a skin tone condition ofinterest. In some embodiments, a first list may represent theup-regulated genes and a second list may represent the down-regulatedgenes of the gene expression signature. The listing(s) may be stored ina data structure of the digital file 30 and the data arranged so that,when the digital file is read by the software application 28, aplurality of character strings are reproduced representing the list ofprobe set IDs. Instead of probe set IDs, equivalent gene names and/orgene symbols (or another nomenclature) may be substituted for a list ofprobe set IDs. Additional data may be stored with the gene expressionsignature and/or the digital file 30 and this is commonly referred to asmetadata, which may include any associated information, for example,cell line or sample source, and microarray identification. Examples oflistings of probe set IDs for a skin hyperpigmentation gene expressionsignature, specifically wherein the skin hyperpigmentation condition isage spots, is set forth in FIG. 2, Tables B (the 200 most up-regulatedgenes) and C (the 200 most down-regulated genes in a skinhyperpigmentation gene expression signature). In some embodiments, oneor more skin hyperpigmentation condition gene expression signatures maybe stored in a plurality of digital files and/or stored on a pluralityof computer readable media. In other embodiments, a plurality of geneexpression signatures (e.g., 32, 34) may be stored in the same digitalfile (e.g., 30) or stored in the same digital file or database thatcomprises the instances 22, 24, and 26.

As previously described, the data stored in the first and second digitalfiles may be stored in a wide variety of data structures and/or formats.In some embodiments, the data is stored in one or more searchabledatabases, such as free databases, commercial databases, or a company'sinternal proprietary database. The database may be provided orstructured according to any model known in the art, such as for exampleand without limitation, a flat model, a hierarchical model, a networkmodel, a relational model, a dimensional model, or an object-orientedmodel. In some embodiments, at least one searchable database is acompany's internal proprietary database. A user of the system 10 may usea graphical user interface associated with a database management systemto access and retrieve data from the one or more databases or other datasources to which the system is operably connected. In some embodiments,the first digital file 20 is provided in the form of a first databaseand the second digital file 30 is provided in the form of a seconddatabase. In other embodiments, the first and second digital files maybe combined and provided in the form of a single file.

In some embodiments, the first digital file 20 may include data that istransmitted across the communication network 18 from a digital file 36stored on the computer readable medium 38. In one embodiment, the firstdigital file 20 may comprise gene expression data obtained from a cellline (e.g., a fibroblast cell line and/or a keratinocyte cell line) aswell as data from the digital file 36, such as gene expression data fromother cell lines or cell types, gene expression signatures, perturbageninformation, clinical trial data, scientific literature, chemicaldatabases, pharmaceutical databases, and other such data and metadata.The digital file 36 may be provided in the form of a database, includingbut not limited to Sigma-Aldrich LOPAC collection, Broad Institute C-MAPcollection, GEO collection, and Chemical Abstracts Service (CAS)databases.

The computer readable medium 16 (or another computer readable media,such as 16) may also have stored thereon one or more digital files 28comprising computer readable instructions or software for reading,writing to, or otherwise managing and/or accessing the digital files 20,30. The computer readable medium 16 may also comprise software orcomputer readable and/or executable instructions that cause thecomputing device 12 to perform one or more steps of the methods of thepresent invention, including for example and without limitation, thestep(s) associated with comparing a gene expression signature stored indigital file 30 to instances 22, 24, and 26 stored in digital file 20.In some embodiments, the one or more digital files 28 may form part of adatabase management system for managing the digital files 20, 28.Non-limiting examples of database management systems are described inU.S. Pat. Nos. 4,967,341 and 5,297,279.

The computer readable medium 16 may form part of or otherwise beconnected to the computing device 12. The computing device 12 can beprovided in a wide variety of forms, including but not limited to anygeneral or special purpose computer such as a server, a desktopcomputer, a laptop computer, a tower computer, a microcomputer, a minicomputer, and a mainframe computer. While various computing devices maybe suitable for use with the present invention, a generic computingdevice 12 is illustrated in FIG. 5. The computing device 12 may compriseone or more components selected from a processor 40, system memory 42,and a system bus 44. The system bus 44 provides an interface for systemcomponents including but not limited to the system memory 42 andprocessor 40. The system bus 36 can be any of several types of busstructures that may further interconnect to a memory bus (with orwithout a memory controller), a peripheral bus, and a local bus usingany of a variety of commercially available bus architectures. Examplesof a local bus include an industrial standard architecture (USA) bus, amicrochannel architecture (MSA) bus, an extended ISA (EISA) bus, aperipheral component interconnect (PCI) bus, a universal serial (USB)bus, and a small computer systems interface (SCSI) bus. The processor 40may be selected from any suitable processor, including but not limitedto, dual microprocessor and other multi-processor architectures. Theprocessor executes a set of stored instructions associated with one ormore program applications or software.

The system memory 42 can include non-volatile memory 46 (e.g., read onlymemory (ROM), erasable programmable read only memory (EPROM),electrically erasable programmable read only memory (EEPROM), etc.)and/or volatile memory 48 (e.g., random access memory (RAM)). A basicinput/output system (BIOS) can be stored in the non-volatile memory 38,and can include the basic routines that help to transfer informationbetween elements within the computing device 12. The volatile memory 48can also include a high-speed RAM such as static RAM for caching data.

The computing device 12 may further include a storage 44, which maycomprise, for example, an internal hard disk drive [HDD, e.g., enhancedintegrated drive electronics (EIDE) or serial advanced technologyattachment (SATA)] for storage. The computing device 12 may furtherinclude an optical disk drive 46 (e.g., for reading a CD-ROM or DVD-ROM48). The drives and associated computer-readable media providenon-volatile storage of data, data structures and the data architectureof the present invention, computer-executable instructions, and soforth. For the computing device 12, the drives and media accommodate thestorage of any data in a suitable digital format. Although thedescription of computer-readable media above refers to an HDD andoptical media such as a CD-ROM or DVD-ROM, it should be appreciated bythose skilled in the art that other types of media which are readable bya computer, such as Zip disks, magnetic cassettes, flash memory cards,cartridges, and the like may also be used, and further, that any suchmedia may contain computer-executable instructions for performing themethods of the present invention.

A number of software applications can be stored on the drives 44 andvolatile memory 48, including an operating system and one or moresoftware applications, which implement, in whole or part, thefunctionality and/or methods described herein. It is to be appreciatedthat the embodiments can be implemented with various commerciallyavailable operating systems or combinations of operating systems. Thecentral processing unit 40, in conjunction with the softwareapplications in the volatile memory 48, may serve as a control systemfor the computing device 12 that is configured to, or adapted to,implement the functionality described herein.

A user may be able to enter commands and information into the computingdevice 12 through one or more wired or wireless input devices 50, forexample, a keyboard, a pointing device, such as a mouse (notillustrated), or a touch screen. These and other input devices are oftenconnected to the central processing unit 40 through an input deviceinterface 52 that is coupled to the system bus 44 but can be connectedby other interfaces, such as a parallel port, an IEEE 1394 serial port,a game port, a universal serial bus (USB) port, an IR interface, etc.The computing device 12 may drive a separate or integral display device54, which may also be connected to the system bus 44 via an interface,such as a video port 56.

The computing devices 12, 14 may operate in a networked environmentacross network 18 using a wired and/or wireless network communicationsinterface 58. The network interface port 58 can facilitate wired and/orwireless communications. The network interface port can be part of anetwork interface card, network interface controller (NIC), networkadapter, or LAN adapter. The communication network 18 can be a wide areanetwork (WAN) such as the Internet, or a local area network (LAN). Thecommunication network 18 can comprise a fiber optic network, atwisted-pair network, a Tl/El line-based network or other links of theT-carrier/E carrier protocol, or a wireless local area or wide areanetwork (operating through multiple protocols such as ultra-mobile band(UMB), long term evolution (LTE), etc.). Additionally, communicationnetwork 18 can comprise base stations for wireless communications, whichinclude transceivers, associated electronic devices formodulation/demodulation, and switches and ports to connect to a backbonenetwork for backhaul communication such as in the case ofpacket-switched communications.

II. Methods for Creating a Plurality of Instances

In some embodiments, the methods of the present invention may comprisepopulating at least the first digital file 20 with a plurality ofinstances (e.g., 22, 24, 26) comprising data derived from a plurality ofgene expression profiling experiments, wherein one or more of theexperiments comprise exposing, for example, keratinocyte cells (or otherskin cells such as human skin equivalent cultures or ex vivo culturedhuman skin) to at least one perturbagen. For simplicity of discussion,the gene expression profiling discussed hereafter will be in the contextof a microarray experiment.

Referring to FIG. 6, one embodiment of a method of the present inventionis illustrated. The method 58 comprises exposing a keratinocyte cell toa perturbagen 64. The perturbagen may be dissolved in a carrier, such asdimethyl sulfoxide (DMSO). After exposure, mRNA is extracted from thecells exposed to the perturbagen and reference cells 66 (e.g.,keratinocyte cells) which are exposed to only the carrier. The mRNA 68,70, 72 may be reverse transcribed to cDNA 64, 76, 78 and marked withdifferent fluorescent dyes (e.g., red and green) if a two colormicroarray analysis is to be performed. Alternatively, the samples maybe prepped for a one color microarray analysis, and further a pluralityof replicates may be processed if desired. The cDNA samples may beco-hybridized to the microarray 80 comprising a plurality of probes 82.The microarray may comprise thousands of probes 82. In some embodiments,there are between 10,000 and 50,000 gene probes 82 present on themicroarray 80. The microarray is scanned by a scanner 84, which excitesthe dyes and measures the amount fluorescence. A computing device 86 maybe used to analyze the raw images to determine the expression levels ofa gene in the cells 60, 62 relative to the reference cells 66. Thescanner 84 may incorporate the functionality of the computing device 86.The expression levels include: i) up-regulation [e.g., greater bindingof the test material (e.g., cDNA 74, 76) to the probe than the referencematerial (e.g., cDNA 78)], or ii) down-regulation [e.g., greater bindingof the reference material (e.g., cDNA 78) to the probe than the testmaterial (e.g., cDNA 74, 76)], iii) expressed but not differentially[e.g., similar binding of the reference material (e.g., cDNA 78) to theprobe than the test material (e.g., cDNA 74. 76)], and iv) no detectablesignal or noise. The up- and down-regulated genes are referred to asdifferentially expressed. Microarrays and microarray analysis techniquesare well known in the art, and it is contemplated that other microarraytechniques may be used with the methods, devices and systems of thepresent invention. For example, any suitable commercial ornon-commercial microarray technology and associated techniques may used.Good results have been obtained with Affymetrix GeneChip® technology andIllumina BeadChip™ technology. One illustrative technique is describedin the Examples, “Generally Applicable” methods section. However, one ofskill in the art will appreciate that the present invention is notlimited to the methodology of the example and that other methods andtechniques are also contemplated to be within its scope.

In a very specific embodiment, an instance consists of the rank ordereddata for all of the probe sets on the Affymetrix HG-U133A2.0 GeneChipwherein each probe on the chip has a unique probe set Identifier. Theprobe sets are rank ordered by the fold change relative to the controlsin the same C-map batch (single instance/average of controls). The probeset Identifiers are rank-ordered to reflect the most up-regulated to themost down-regulated.

Notably, even for the non-differentially regulated genes the signalvalues for a particular probe set are unlikely to be identical for theinstance and control so a fold change different from 1 will becalculated that can be used for comprehensive rank ordering. Inaccordance with methods disclosed by Lamb et al. (2006), data areadjusted using 2 thresholds to minimize the effects of genes that mayhave very low noisy signal values, which can lead to spurious large foldchanges. The thresholding is preferably done before the rank ordering.An example for illustrative purposes includes a process wherein a firstthreshold is set at 20. If the signal for a probe set is below 20, it isadjusted to 20. Ties for ranking are broken with a second thresholdwherein the fold changes are recalculated and any values less than 2 areset to 2. For any remaining ties the order depends on the specificsorting algorithm used but is essentially random. The probe sets in themiddle of the list do not meaningfully contribute to an actualconnectivity score.

The rank ordered data are stored as an instance. The probes may besorted into a list according to the level of gene expression regulationdetected, wherein the list progresses from up-regulated to marginal orno regulation to down-regulated, and this rank ordered listing of probeIDs is stored as an instance (e.g., 22) in the first digital file 20.Referring to FIG. 4, the data associated with an instance comprises theprobe ID 80 and a value 82 representing its ranking in the list (e.g.,1, 2, 3, 4 . . . N, where N represents the total number of probes on themicroarray). The ordered list 84 may generally comprise approximatelythree groupings of probe IDs: a first grouping 86 of probe IDsassociated with up-regulated genes, a second group 88 of probe IDsassociated with genes with marginal regulation or no detectable signalor noise, and a third group 90 of probe IDs associated withdown-regulated genes. The most up regulated genes are at or near the topof the list 84 and the most down-regulated genes are at or near thebottom of the list 84. The groupings are shown for illustration, but thelists for each instance may be continuous and the number of regulatedgenes will depend on the strength of the effect of the perturbagenassociated with the instance. Other arrangements within the list 84 maybe provided. For example, the probe IDs associated with thedown-regulated genes may be arranged at the top of the list 84. Thisinstance data may also further comprise metadata such as perturbagenidentification, perturbagen concentration, cell line or sample source,and microarray identification.

In some embodiments, one or more instances comprise at least about1,000, 2,500, 5,000, 10,000, or 20,000 identifiers and/or less thanabout 30,000, 25,000, or 20,000 identifiers. In some embodiments, thedatabase comprises at least about 50, 100, 250, 500, or 1,000 instancesand/or less than about 50,000, 20,000, 15,000, 10,000, 7,500, 5,000, or2,500 instances. Replicates of an instance may be created, and the sameperturbagen may be used to derive a first instance from keratinocytecells and a second instance from another skin cell type, such asfibroblasts, melanocytes, melanoma or complex tissue, for example exvivo human skin.

The present inventors have discovered that instances derived with a celltype, such as keratinocyte cells, are more predictive than other celltypes when used in combination with a skin lightening benchmark agentexpression signature derived from the same cell type. In other words,better results are achieved if the cell type used to generate the querysignature is the same as the instance cell type. While thiscell-consistency guide may appear predictable, what is surprising isthat with respect to benchmark agent signatures the present inventorssurprisingly discovered that certain benchmark skin active agents havegreater predictive efficacy with certain cell types over others. Forexample, as set forth in Examples 4 and 5, the present inventorscompared Retinoic Acid benchmark gene expression signatures derived fromBJ fibroblast cells and keratinocyte cells, it was surprisinglydiscovered that those derived from keratinocyte cells yield betterresults with respect to a potential agent hit list based on connectivitywith the query signature.

III. Methods for Deriving Hyperpigmentation Gene Expression Signatures

Some methods of the present invention comprise identifying a geneexpression signature that represents the up-regulated and down-regulatedgenes associated with a skin condition of interest, in particular withskin tone or hyperpigmentation.

The pathogenesis of a skin pigmentation condition typically involvescomplex processes involving numerous known and unknown extrinsic andintrinsic factors, as well as responses to such factors that are subtleover a relatively short period of time but non-subtle over a longerperiod of time. This is in contrast to what is typically observed indrug development and drug screening methods, wherein a specific target,gene, or mechanism of action is of interest. Due to the unique screeningchallenges associated with a skin pigmentation condition, the quality ofthe gene expression signature representing the condition of interest canbe important for distinguishing between the gene expression dataactually associated with a response to a perturbagen from the backgroundexpression data.

One challenge in developing gene expression signatures for skin tone andpigmentation-related skin disorders is that the number of genes selectedneeds to be adequate to reflect the dominant and key biology but not solarge as to include many genes that have achieved a level of statisticalsignificance by random chance and are non-informative. Thus, querysignatures should be carefully derived since the predictive value may bedependent upon the quality of the gene expression signature.

One factor that can impact the quality of the query signature is thenumber of genes included in the signature. The present inventors havefound that, with respect to a cosmetic data architecture andconnectivity map, too few genes can result in a signature that isunstable with regard to the highest scoring instances. In other words,small changes to the gene expression signature can result in significantdifferences in the highest scoring instance. Conversely, too many genesmay tend to partially mask the dominant biological responses and willinclude a higher fraction of genes meeting statistical cutoffs by randomchance—thereby adding undesirable noise to the signature. The inventorshave found that the number of genes desirable in a gene expressionsignature is also a function of the strength of the biological responseassociated with the condition and/or the number of genes needed to meetminimal values (e.g., a p-value less than about 0.05 or less than about1.0, or in accordance with applicable statistical principles) forstatistical significance. Hence, what is considered an ideal number ofgenes will vary from condition to condition. When the biology is weaker,such as is the case typically with cosmetic condition phenotypes, fewergenes than those which may meet the statistical requisite for inclusionin the prior art, may be used to avoid adding noisy genes.

For example, the present inventors have determined that where geneexpression profiling analysis of a skin condition yields from betweenabout 2,000 and 4,000 genes having a statistical p-value of less than0.05 and approximately 1000 genes having a p-value of less than 0.001, avery strong biological response is indicated. A moderately strongbiological response may yield approximately 800-2000 genes have astatistical p-value of less than 0.05 combined with approximately400-600 genes have a p-value of less than 0.001. In these cases, a geneexpression signature comprising between about 100 and about 600 genesappears ideal. Weaker biology may be better represented by a geneexpression signature comprising fewer genes, such as between about 20and 100 genes.

While a gene expression signature may represent all significantlyregulated genes associated with a skin condition of interest; typicallyit represents a subset of such genes. The present inventors havediscovered that hyperpigmentation gene expression signatures comprisingbetween about 50 and about 400 genes of approximately equal numbers ofup-regulated and/or down-regulated genes are stable, reliable, and canprovide predictive results. For example, a suitable gene expressionsignature may have from about 100-150 genes, 250-300 genes, 300-350genes, or 350-400 genes. In a very specific embodiment, ahyperpigmentation gene expression signature includes the 100 most up-and down-regulated genes. However, one of skill in the art willappreciate that gene expression signatures comprising fewer or moregenes are also within the scope of the various embodiments of theinvention. For purposes of depicting a gene expression signature, theprobe set IDs associated with the genes are preferably separated into afirst list comprising the most up-regulated genes and a second listcomprising the most down-regulated, as set forth in FIG. 2, Tables B andC.

Gene expression signatures may be generated from full thickness skinbiopsies from skin having the skin condition of interest compared to acontrol. For generation of an exemplary hyperpigmentation geneexpression signature, biopsies are taken from forearm age spots andcompared to non-affected forearm skin sampled from the same subject.

In other embodiments of the present invention, a gene expressionsignature may be derived from a gene expression profiling analysis ofkeratinocyte, melanocyte, fibroblast or melanoma cells treated with oneor more benchmark skin-active agents, in particular a skin-lighteningagent, to represent cellular perturbations leading to improvement in theskin tissue condition treated with that benchmark skin active agent,wherein the signature comprises a plurality of genes up-regulated anddown-regulated by the benchmark skin active agent in cells in vitro. Asone illustrative example, microarray gene expression profile data wherethe perturbagen is the known skin lightening agent Niacinamide may beanalyzed using the present invention to determine from the rank-orderedinstances in the query results, the perturbagens associated with thehighest scoring instances.

A composite benchmark signature according to the invention is asignature derived from a cell treated with more than one benchmark skinactive agent (described in Examples 3, 6, and 7). The actives may beselected to reflect more than one mechanism of action in skin, or may beselected to reflect more than one attribute of general skin tone.

In a further specific embodiment, a benchmark skin-lightening geneexpression signature is compared to a benchmark skin-darkening geneexpression signature and genes not differentially regulated between thetwo are eliminated from the signature intended to be the querysignature. Non-limiting examples of skin-darkening agents according tothis embodiment include alpha-melanocyte-stimulating hormone (a-MSH) andany related melanocortin 1 receptor agonists or stimulant thereof.

IV. Methods for Comparing a Plurality of Instances to One or MorePigmentation Gene Expression Signatures

Referring to FIG. 7 and FIG. 8, a method for querying a plurality ofinstances with one or more hyperpigmentation-relevant gene signatureswill now be described. Broadly, the method comprises querying aplurality of instances with one or more hyperpigmentation-relevant genesignatures and applying a statistical method to determine how stronglythe signature genes match the regulated genes in an instance. Positiveconnectivity occurs when the genes in the up-regulated signature listare enriched among the up-regulated genes in an instance and the genesin the down-regulated signature list are enriched among thedown-regulated genes in an instance. On the other hand, if theup-regulated genes of the signature are predominantly found among thedown-regulated genes of the instance, and vice versa, this is scored asnegative connectivity. FIG. 7 schematically illustrates an extremeexample of a positive connectivity between signature 90 and the instance104 comprising the probe IDs 102, wherein the probe IDs of the instanceare ordered from most up-regulated to most down-regulated. In thisexample, the probe IDs 100 (e.g., X₁, X₂ X₃, X₄, X₅, X₆, X₇, X₈) of thegene signature 90, comprising an up list 97 and a down list 99, have aone to one positive correspondence with the most up-regulated anddown-regulated probe IDs 102 of the instance 104, respectively.Similarly, FIG. 8 schematically illustrates an extreme example of anegative connectivity between signature 94 and the instance 88comprising the probe IDs 90, wherein the probe IDs of the instance areordered from most up-regulated to most down-regulated. In this example,the probe IDs of the up list 93 (e.g., X₁, X₂ X₃, X₄) correspond exactlywith the most down-regulated genes of the instance 88, and the probe IDsof the down list 95 (e.g., X₅, X₆, X₇, X₈) correspond exactly to themost up-regulated probe IDs of the instance 88. FIG. 9 schematicallyillustrates an extreme example of neutral connectivity, wherein there isno consistent enrichment of the up- and down-regulated genes of thesignature among the up- and down-regulated genes of the instance, eitherpositive or negative. Hence the probe IDs 106 (e.g., X₁, X₂ X₃, X₄, X₅,X₆, X₇, X₈) of a gene signature 108 (comprising an up list 107 and adown list 109) are scattered with respect to rank with the probe IDs 110of the instance 112, wherein the probe IDs of the instance are orderedfrom most up-regulated to most down-regulated. While the aboveembodiments illustrate process where the gene signature comprises a bothan up list and a down list representative of the most significantly up-and down-regulated genes of a skin condition, it is contemplated thatthe gene signature may comprise only an up list or a down list when thedominant biology associated with a condition of interest shows generegulation in predominantly one direction.

In some embodiments, the connectivity score can be a combination of anup-score and a down score, wherein the up-score represents thecorrelation between the up-regulated genes of a gene signature and aninstance and the down-score represents the correlation between thedown-regulated genes of a gene signature and an instance. The up scoreand down score may have values between +1 and −1. For an up score (anddown score) a high positive value indicates that the correspondingperturbagen of an instance induced the expression of the microarrayprobes of the up-regulated (or down-regulated) genes of the genesignature, and a high negative value indicates that the correspondingperturbagen associated with the instance repressed the expression of themicroarray probes of the up-regulated (or down-regulated) genes of thegene signature. The up-score can be calculated by comparing eachidentifier of an up list of a gene signature comprising the up-regulatedgenes (e.g., Tables B, D, F, H and lists 93, 97, and 107) to an orderedinstance list, while the down-score can be calculated by comparing eachidentifier of a down list of a gene signature comprising thedown-regulated genes (see, e.g., Tables C, E, G, I and down lists 95,99, and 109) to an ordered instance list. In these embodiments, the genesignature comprises the combination of the up list and the down list.

In some embodiments, the connectivity score value may range from +2(greatest positive connectivity) to −2 (greatest negative connectivity),wherein the connectivity score (e.g., 101, 103, and 105) is thecombination of the up score (e.g., 111, 113, 115) and the down score(e.g., 117, 119, 121) derived by comparing each identifier of a genesignature to the identifiers of an ordered instance list. In otherembodiments the connectivity range may be between +1 and −1. Examples ofthe scores are illustrated in FIGS. 7, 8 and 9 as reference numerals101, 103, 105, 111, 113, 115, 117, 119, and 121. The strength ofmatching between a signature and an instance represented by the upscores and down scores and/or the connectivity score may be derived byone or more approaches known in the art and include, but are not limitedto, parametric and non-parametric approaches. Examples of parametricapproaches include Pearson correlation (or Pearson r) and cosinecorrelation. Examples of non-parametric approaches include Spearman'sRank (or rank-order) correlation, Kendall's Tau correlation, and theGamma statistic. Generally, in order to eliminate a requirement that allprofiles be generated on the same microarray platform, a non-parametric,rank-based pattern matching strategy based on the Kolmogorov-Smirnovstatistic (see M. Hollander et al. “Nonparametric Statistical Methods”;Wiley, New York, ed. 2, 1999)(see, e.g., pp. 178-185). It is noted,however, that where all expression profiles are derived from a singletechnology platform, similar results may be obtained using conventionalmeasures of correlation, for example, the Pearson correlationcoefficient.

In specific embodiments, the methods and systems of the presentinvention employ the nonparametric, rank-based pattern-matching strategybased on the Kolmogorov-Smirnov statistic, which has been refined forgene profiling data by Lamb's group, commonly known in the art as GeneSet Enrichment Analysis (GSEA) (see, e.g., Lamb et al. 2006 andSubramanian, A. et al. (2005) Proc. Natl. Acad Sci U.S.A., 102,15545-15550). For each instance, a down score is calculated to reflectthe match between the down-regulated genes of the query and theinstance, and an up score is calculated to reflect the correlationbetween the up-regulated genes of the query and the instance. In certainembodiments the down score and up score each may range between −1 and+1. The combination represents the strength of the overall match betweenthe query signature and the instance.

The combination of the up score and down score is used to calculate anoverall connectivity score for each instance, and in embodiments whereup and down score ranges are set between −1 and +1, the connectivityscore ranges from −2 to +2, and represents the strength of match betweena query signature and the instance. The sign of the overall score isdetermined by whether the instance links positivity or negatively to thesignature. Positive connectivity occurs when the perturbagen associatedwith an instance tends to up-regulate the genes in the up list of thesignature and down-regulate the genes in the down list. Conversely,negative connectivity occurs when the perturbagen tends to reverse theup and down signature gene expression changes, The magnitude of theconnectivity score is the sum of the absolute values of the up and downscores when the up and down scores have different signs. A high positiveconnectivity score predicts that the perturbagen will tend to induce thecondition that was used to generate the query signature, and a highnegative connectivity score predicts that the perturbagen will tend toreverse the condition associated with the query signature. A zero scoreis assigned where the up and down scores have the same sign, indicatingthat a perturbagen did not have a consistent impact the conditionsignature (e.g., up-regulating both the up and down lists).

According to Lamb et al. (2006), there is no standard for estimatingstatistical significance of connections observed. Lamb teaches that thepower to detect connections may be greater for compounds with manyreplicates. Replicating in this context means that the same perturbagenis profiled multiple times. Where batch to batch variation must beavoided, a perturbagen should be profiled multiple times in each batch.However, since microarray experiments tend to have strong batch effectsit is desirable to replicate instances in different batches (i.e.,experiments) to have the highest confidence that connectivity scores aremeaningful and reproducible.

Each instance may be rank ordered according to its connectivity score tothe query signature and the resulting rank ordered list displayed to auser using any suitable software and computer hardware allowing forvisualization of data.

In some embodiments, the methods may comprise identifying from thedisplayed rank-ordered list of instances (i) the one or moreperturbagens associated with the instances of interest (therebycorrelating activation or inhibition of a plurality of genes listed inthe query signature to the one or more perturbagens); (ii) thedifferentially expressed genes associated with any instances of interest(thereby correlating such genes with the one or more perturbagens, theskin tissue condition of interest, or both); (iii) the cells associatedwith any instance of interest (thereby correlating such cells with oneor more of the differentially expressed genes, the one or moreperturbagens, and the skin tissue condition of interest); or (iv)combinations thereof. The one or more perturbagens associated with aninstance may be identified from the metadata stored in the database forthat instance. However, one of skill in the art will appreciate thatperturbagen data for an instance may be retrievably stored in and byother means. Because the identified perturbagens statistically correlateto activation or inhibition of genes listed in the query signature, andbecause the query signature is a proxy for a skin condition of interest,e.g. a hyperpigmentation condition, the identified perturbagens may becandidates for new cosmetic agents, new uses of known cosmetic agents,or to validate known agents for known uses relevant to thehyperpigmentation condition.

In some embodiments, the methods of the present invention may furthercomprise testing the selected candidate cosmetic agent, using in vitroassays and/or in vivo testing, to validate the activity of the agent andusefulness as a cosmetic agent. Any suitable in vitro test method can beused, including those known in the art, and most preferably in vitromodels developed in accordance with the present invention. For example,MatTek human skin equivalent cultures or other skin equivalent culturesmay be treated with one or a combination of perturbagens selected formimicry of the skin condition of interest with respect to regulation ofthe genes constituting a physiological theme pattern for the skincondition of interest. In some embodiments, evaluation of selectedagents using in vitro assays may reveal, confirm, or both, that one ormore new candidate cosmetic agents may be used in conjunction with aknown cosmetic agent (or a combination of known cosmetic agents) toregulate a skin condition of interest.

Clinical testing can be useful to confirm putative skin-pigmentationmodifying efficacy. Clinical methods include live expert grading,chromameter, and color image capture and analysis. A new useful clinicalmeasurement tool in assessing effectiveness is based on the principle ofnoncontact SIAscopy™, a recently described method to measure skinmelanin content and distribution. It rapidly captures facial maps ofskin chromophores, permitting determination of the content anddistribution of melanin in any spot or any area of the skin. Clinicaltesting on various body sites such as forearm, face, chest, and backhave been reported, and all have utility in evaluating technology. Anythoroughly controlled clinical evaluation is expensive and thereforepracticality limits testing to only the most promising candidates.

V. Hyperpigmentation Disorders

The present invention provides methods for identifying putative skinactive agents for the treatment of pigmentation conditions anddisorders, and in particular those relating to hyperpigmentation. Mainfactors in the development of conditions of hyperpigmentation areexposure to certain environmental conditions and hormonal changes. Ingeneral, the number of active melanocytes per unit area of skindecreases with age (10-20% decline per decade), and there are moreactive melanocytes in chronically sun-exposed skin than in non-exposedskin. This increased number of active melanocytes in sun-damaged skinindicates the influence of chronic UV exposure (e.g., on face, hands,and arms) in stimulating melanogenic activity. Since chronic UV exposurealso alters dermal fibroblast function in aging skin and sincefibroblasts appear to play a regulatory role in melanin production,dermal damage from sunlight may contribute to the production ofhyperpigmentation in exposed aging skin.

Post-inflammatory hyperpigmentation (PIH) results from inflammation ofthe skin and disproportionately affects people with darker skin.Inflammation induced pigmentation is often seen associated with acnelesions, ingrown hairs, scratches, insect bites, and surfactant damage.As an example of the latter, exposure of human forearm skin to the harshsurfactant sodium lauryl sulfate (SLS) under patch for a few hours willproduce erythema within a day. Over the course of 1-2 weeks after thisSLS exposure, hyperpigmentation will result, particularly in darkerskin, but it will occur even in Caucasian skin. Topical treatment withanti-inflammatory agents is known to ameliorate this. A non-limitingexample is phytosterol.

Exposure of skin to sunlight is the most common cause of skinhyperpigmentation and is increasingly believed that it is a subset ofPIH caused by a post-inflammatory response to UV damage to skin. Theinflammatory response may be the result of an obvious acute inflammatoryevent such as sunburn or due to repeated sub-erythemal exposures to UV.While in the latter, there may not be visible erythema, histologically,such exposed skin has elevated inflammatory cell content, yielding a“subclinical” inflammatory process. This explanation is supported by thefact that topical treatment with anti-inflammatory agents immediatelyafter UVB exposure prevents induction of delayed tanning.

Inflammation may result in hyperpigmentation through several mechanisms.Among them is direct stimulation of melanocytes by inflammatorymediators such as IL-1-alpha. Reactive oxygen species such as superoxideand nitric oxide generated in damaged skin (e.g., from UV exposure) orreleased as by-products from inflammatory cells are also knownstimulators of melanocytes. Additionally, damage induced in epidermalcells can lead to release of endocrine inducers of pigmentation such asalpha-melanin stimulating hormone (MSH). The resulting hyperpigmentationinduced by all these effects is adaptive since it appears to providesome measure of protection against subsequent insult since melanin isknown to have both UV absorption and reactive oxygen species scavengingcapacity.

The melanin produced during an inflammatory event also can enter thedermis where it is engulfed by macrophages, producing “melanophages.”These cells are often retained in the upper dermis for prolonged periodssince removal of dermal melanin apparently is a very slow process. Thus,post-inflammatory hyperpigmentation can be a very long-lived problem forthe skin.

Solar (Actinic) Lentigos are hyperpigmented spots also known aslentigines, age spots, and liver spots. They develop as a result ofchronic exposure of skin to UV radiation and occur on sun-exposed partsof the body (in particular, the hands, arms, face, upper chest, andshoulders). Chronic exposure of skin to UV results in chronicinflammation, such as the epidermal endothelin cascade. The darkappearance of age spots results from excessive melanin in the region,and may result from overproduction of melanin in the hyperactivemelanocytes, longer retention of melanin in aging epidermis due to theslower turnover of this tissue layer, longer retention of melanin inkeratinocytes within rete ridges, and dermal melanin-containingmelanophages, which have been observed histologically to lie beneath thelentigines. There is reduced wound healing with age at least in part dueto reduced clearance of materials from dermis apparently due to vascularand lymphatic changes, so that the residence time of melanophages indermis may be lengthened in older populations.

Certain observations suggest that there is a change in the genetic andphenotypic expression within an age spot as compared to cells insurrounding non-affected skin. For example, within lesional lentigoskin, the rete ridges are greatly exaggerated, extending deeper into thedermis. This deep penetration runs counter to the general observation offlattening of the convoluted dermal-epidermal junction with aging,evidenced by the diminution of the rete ridges. In solar lentigines, thebasement membrane is also perturbed, which likely contributes to melaninentering the dermis to result in melanophage formation.

Moreover, the expression levels of several melanogenesis-associatedgenes are known to be increased in actinic lentigos. There is anaccentuation of the epidermal endothelin inflammatory cascade, togetherwith decreased proliferation and differentiation of lesionalkeratinocytes. Many of these changes appear to be permanent since thesespots persist even when further UV exposure is avoided.

While lentigos appear to be permanent, their melanin content and thustheir intensity will vary seasonally. For example, in evaluation ofwomen with facial hyperpigmented spots in October versus December (inKobe, Japan or Cincinnati, Ohio, USA), there is a marked reduction inthe size of spots over that time period, suggesting that the lack ofcontinued exposure to sunlight in winter leads to gradual reduction inmelanin production (seasonal fading) even in hyperpigmented spots.Additionally, in a separate examination of facial spots in March versusMay (in Cincinnati, Ohio, USA), a marked increase in the size of spotsis observed, consistent with the expected increased pigmentation due toincreased sun exposure in spring (seasonal darkening).

From a consumer appearance standpoint, hyperpigmented spots and unevenpigmentation are important in the perception of age. In a series ofstudies, facial images were digitally modified to remove allage-defining textural features (e.g., facial furrows, folds, lines,wrinkles) leaving only pigmentation as the variable. Studies have shownthat when using naïve judge evaluation and computer image analysis ofthe facial images, pigmentation features can contribute to up to 20years in perceived age of individuals.

The hyperpigmentary disorder generally referred to as melasma is notwell understood. It occurs typically as symmetrical lesions on the face,primarily in darker skin type females at puberty or later in life.Sunlight exposure is likely a factor in the development of melasma sinceit occurs on the face (a sun-exposed body site) and since the conditionworsens in the summer. Most melasma sufferers have a hypersensitivity toultraviolet radiation, and even brief exposures to sunlight canstimulate hyperpigmentation. There is also a hormonal component, likelyprogesterone, since episodes of melasma are often associated withpregnancy and the use of hormonal birth control. There may also be anestrogen component since estrogen receptor expression is increased inmelasma.

In melasma lesions, there is excess melanin present in both theepidermis and upper dermis, associated with extravascular macrophages.Since there is only a slight increase in the number of melanocytes, theabnormality appears to be in function of the skin cells, in particular,increase expression of factors in keratinocytes, fibroblasts, andmelanocytes of the involved skin. In contrast to PIH, there is noapparent inflammatory phase involved in its development. Additionally,there is likely a genetic compound predisposing individuals to melasma,although the specific genetic basis for it is not defined.

The pigmentation process is complex as evidenced particularly by recentrevelations from genomic and proteomic analysis. There are approximately1,500 gene products (proteins) expressed in melanosomes of alldevelopmental stages, with 600 of them being expressed at any giventime, and with 100 of them apparently unique to the melanosome. Added tothis are many other proteins (membrane-associated, cytoskeletal,transport, etc.) involved in pigmentation in both the melanocyte and thekeratinocyte, indicating the complexity of the pigmentary process. Whilethe basic process (e.g., stimulation of melanocytes and conversion oftyrosine to melanin) is well studied, there are many regulatory elementsthat have emerged from recent research involved in signaling, in thetransport of melanosomes within the melanocyte, and the transfer ofmelanosomes to the keratinocyte.

VI. Cosmetic Compositions and Personal Care Products

Generally, skin-active agents identified for the enhancement of skintone or for treatment of pigment-related skin conditions may be appliedin accordance with cosmetic compositions and formulation parameterswell-known in the art. Various methods of treatment, application,regulation, or improvement may utilize the skin care compositionscomprising skin-active agents identified according to the inventivemethods.

Skin hyperpigmentation as a cosmetic concern is generally treated bytopical formulation administration, so that depigmentation is restrictedto hyperpigmented areas and normal skin is left unaffected by the drug.

Because of the desirability of providing various cosmetic skinanti-aging benefits to a consumer, it may be beneficial to incorporatetest agents or compounds identified by one or more of the screeningmethods described herein into a cosmetic composition suitable fortopical application to skin. That is, it may be desirable to include thetest agent as an ingredient in the cosmetic composition. In certainembodiments, the cosmetic composition may include a dermatologicalacceptable carrier, the test agent, and one or more optional ingredientsof the kind commonly included in the particular cosmetic compositingbeing provided.

Dermatologically acceptable carriers should be safe for use in contactwith human skin tissue. Suitable carriers may include water and/or watermiscible solvents. The cosmetic skin care composition may comprise fromabout 1% to about 95% by weight of water and/or water miscible solvent.The composition may comprise from about 1%, 3%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% to about90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, or 5% water and/or water miscible solvents. Suitablewater miscible solvents include monohydric alcohols, dihydric alcohols,polyhydric alcohols, glycerol, glycols, polyalkylene glycols such aspolyethylene glycol, and mixtures thereof. When the skin carecomposition is in the form of an emulsion, water and/or water misciblesolvents are carriers typically associated with the aqueous phase.

Suitable carriers also include oils. The skin care composition maycomprise from about 1% to about 95% by weight of one or more oils. Thecomposition may comprise from about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, 5%, or 3% of one or more oils. Oils may be usedto solubilize, disperse, or carry materials that are not suitable forwater or water soluble solvents. Suitable oils include silicones,hydrocarbons, esters, amides, ethers, and mixtures thereof. The oils maybe volatile or nonvolatile.

Suitable silicone oils include polysiloxanes. Commercially availablepolysiloxanes include the polydimethylsiloxanes, which are also known asdimethicones, examples of which include the DM-Fluid series fromShin-Etsu, the Vicasil® series sold by Momentive Performance MaterialsInc., and the Dow Corning® 200 series sold by Dow Corning Corporation.Specific examples of suitable polydimethylsiloxanes include Dow Corning®200 fluids (also sold as Xiameter® PMX-200 Silicone Fluids) havingviscosities of 0.65, 1.5, 50, 100, 350, 10,000, 12,500 100,000, and300,000 centistokes.

Suitable hydrocarbon oils include straight, branched, or cyclic alkanesand alkenes. The chain length may be selected based on desiredfunctional characteristics such as volatility. Suitable volatilehydrocarbons may have between 5-20 carbon atoms or, alternately, between8-16 carbon atoms.

Other suitable oils include esters. The suitable esters typicallycontained at least 10 carbon atoms. These esters include esters withhydrocarbyl chains derived from fatty acids or alcohols (e.g.,mono-esters, polyhydric alcohol esters, and di- and tri-carboxylic acidesters). The hydrocarbyl radicals of the esters hereof may include orhave covalently bonded thereto other compatible functionalities, such asamides and alkoxy moieties (e.g., ethoxy or ether linkages, etc.).

Other suitable oils include amides. Amides include compounds having anamide functional group while being liquid at 25° C. and insoluble inwater. Suitable amides include N-acetyl-N-butylaminopropionate,isopropyl N-lauroylsarcosinate, and N,N,-diethyltoluamide. Othersuitable amides are disclosed in U.S. Pat. No. 6,872,401.

Other suitable oils include ethers. Suitable ethers include saturatedand unsaturated fatty ethers of a polyhydric alcohol, and alkoxylatedderivatives thereof. Exemplary ethers include C₄₋₂₀ alkyl ethers ofpolypropylene glycols, and di-C₈₋₃₀ alkyl ethers. Suitable examples ofthese materials include PPG-14 butyl ether, PPG-15 stearyl ether,dioctyl ether, dodecyl octyl ether, and mixtures thereof.

The skin care composition may comprise an emulsifier. An emulsifier isparticularly suitable when the composition is in the form of an emulsionor if immiscible materials are being combined. The skin care compositionmay comprise from about 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, or 1% to about20%, 10%, 5%, 3%, 2%, or 1% emulsifier. Emulsifiers may be nonionic,anionic or cationic. Non-limiting examples of emulsifiers are disclosedin U.S. Pat. Nos. 3,755,560, 4,421,769, and McCutcheon's, Emulsifiersand Detergents, 2010 Annual Ed., published by M. C. Publishing Co. Othersuitable emulsifiers are further described in the Personal Care ProductCouncil's International Cosmetic Ingredient Dictionary and Handbook,Thirteenth Edition, 2006, under the functional category of“Surfactants—Emulsifying Agents.”

Linear or branched type silicone emulsifiers may also be used.Particularly useful polyether modified silicones include KF-6011,KF-6012, KF-6013, KF-6015, KF-6015, KF-6017, KF-6043, KF-6028, andKF-6038 from Shin Etsu. Also particularly useful are thepolyglycerolated linear or branched siloxane emulsifiers includingKF-6100, KF-6104, and KF-6105 from Shin Etsu. Emulsifiers also includeemulsifying silicone elastomers. Suitable silicone elastomers may be inthe powder form, or dispersed or solubilized in solvents such asvolatile or nonvolatile silicones, or silicone compatible vehicles suchas paraffinic hydrocarbons or esters. Suitable emulsifying siliconeelastomers may include at least one polyalkyl ether or polyglycerolatedunit.

Structuring agents may be used to increase viscosity, thicken, solidify,or provide solid or crystalline structure to the skin care composition.Structuring agents are typically grouped based on solubility,dispersibility, or phase compatibility. Examples of aqueous or waterstructuring agents include polymeric agents, natural or synthetic gums,polysaccharides, and the like. In one embodiment, the composition maycomprises from about 0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%,3%, 5% to about 25%, 20%, 10%, 7%, 5%, 4%, or 2%, by weight of thecomposition, of one or more structuring agents.

Polysaccharides and gums may be suitable aqueous phase thickeningagents. Suitable classes of polymeric structuring agents include but arenot limited to carboxylic acid polymers, polyacrylamide polymers,sulfonated polymers, high molecular weight polyalkylglycols orpolyglycerins, copolymers thereof, hydrophobically modified derivativesthereof, and mixtures thereof. Silicone gums are another oil phasestructuring agent. Another type of oily phase structuring agent includessilicone waxes. Silicone waxes may be referred to as alkyl siliconewaxes which and are semi-solids or solids at room temperature. Other oilphase structuring agents may be one or more natural or synthetic waxessuch as animal, vegetable, or mineral waxes.

The skin care compositions may be generally prepared by conventionalmethods such as known in the art of making compositions and topicalcompositions. Such methods typically involve mixing of ingredients in ormore steps to a relatively uniform state, with or without heating,cooling, application of vacuum, and the like. Typically, emulsions areprepared by first mixing the aqueous phase materials separately from thefatty phase materials and then combining the two phases as appropriateto yield the desired continuous phase. The compositions are preferablyprepared such as to optimize stability (physical stability, chemicalstability, photostability, etc.) and/or delivery of active materials.The composition may be provided in a package sized to store a sufficientamount of the composition for a treatment period. The size, shape, anddesign of the package may vary widely. Certain package examples aredescribed in USPNs D570,707; D391,162; D516,436; D535,191; D542,660;D547,193; D547,661; D558,591; D563,221; 2009/0017080; 2007/0205226; and2007/0040306.

EXAMPLES

The present invention will be better understood by reference to thefollowing examples which are offered by way of illustration notlimitation.

Generally Applicable C-Map Methodology

Generating Instances

Individual experiments (referred to as batches) generally comprise 30 to96 samples analyzed using Affymetrix GeneChip® technology platforms,containing 6 replicates of the vehicle control (e.g., DSMO), 2 replicatesamples of a positive control that gives a strong reproducible effect inthe cell type used, and samples of the test material/perturbagen.Replication of the test material is done in separate batches due tobatch effects. In vitro testing was performed in 6-well plates toprovide sufficient RNA for GeneChip® analysis (2-4 μg total RNAyield/well).

Human telomerized keratinocytes (tKC) were obtained from the Universityof Texas, Southwestern Medical Center, Dallas, Tex. tKC cells were grownin EpiLife® media with 1× Human Keratinocyte Growth Supplement(Invitrogen, Carlsbad, Calif.) on collagen I coated cell culture flasksand plates (Becton Dickinson, Franklin Lakes, N.J.). Keratinocytes wereseeded into 6-well plates at 20,000 cells/cm² 24 hours before chemicalexposure. Human skin fibroblasts (BJ cell line from ATCC, Manassas, Va.)were grown in Eagle's Minimal Essential Medium (ATCC) supplemented with10% fetal bovine serum (HyClone, Logan, Utah) in normal cell cultureflasks and plates (Corning, Lowell, Mass.). BJ fibroblasts were seededinto 6-well plates at 12,000 cells/cm² 24 hours before chemicalexposure.

All cells were incubated at 37° C. in a humidified incubator with 5%CO₂. At t=−24 hours cells were trypsinized from T-75 flasks and platedinto 6-well plates in basal growth medium. At t=0 media was removed andreplaced with the appropriate dosing solution as per the experimentaldesign. Dosing solutions were prepared the previous day in sterile 4 mlFalcon snap cap tubes. Pure test materials may be prepared at aconcentration of 1-200 μM, and botanical extracts may be prepared at aconcentration of 0.001 to 1% by weight of the dosing solution. After 6to 24 hours of chemical exposure, cells were viewed and imaged. Thewells were examined with a microscope before cell lysis and RNAisolation to evaluate for morphologic evidence of toxicity. Ifmorphological changes were sufficient to suggest cytotoxicity, a lowerconcentration of the perturbagen was tested. Cells were then lysed with350 ul/well of RLT buffer containing β-mercaptoethanol (Qiagen,Valencia, Calif.), transferred to a 96-well plate, and stored at −20° C.

RNA from cell culture batches was isolated from the RLT buffer usingAgencourt® RNAdvance Tissue-Bind magnetic beads (Beckman Coulter)according to manufacturer's instructions. 1 μg of total RNA per samplewas labeled using Ambion Message Amp™ II Biotin Enhanced kit (AppliedBiosystems Incorporated) according to manufacturer's instructions. Theresultant biotin labeled and fragmented cRNA was hybridized to anAffymetrix HG-U133A 2.0 GeneChip®, which was then washed, stained andscanned using the protocol provided by Affymetrix.

Example 1

This Example illustrates use of C-map to identify connections betweenperturbens and genes associated with a pigmentation condition, whereinthe pigmentation condition is a hyperpigmentation condition.Specifically an analysis of tissue from the arms of individuals showingthe hyperpigmentation condition Solar Lentigines (age spots) is comparedto an analysis of tissue from full normal controls, and an expressionsignature is created as herein described through specific statisticalcomparisons, filtering, and sorting. The expression signature can thenbe used to implement a data architecture by providing a C-map query toidentify relationships between perturbens and genes associated with ahyperpigmentation pigmentation condition.

Deriving a Hyperpigmentation Condition Expression Signature

RNA isolated from clinical samples was analyzed using the AffymetrixHG-U133 Plus 2.0 GeneChips, which contain 54,613 probe setscomplementary to the transcripts of more than 20,000 genes. However,instances in the provided database used were derived from geneexpression profiling experiments using Affymetrix HG-U133A 2.0GeneChips, containing 22,214 probe sets, which are a subset of thosepresent on the Plus 2.0 GeneChip. Therefore, in developing geneexpression signatures from the clinical data, the probe sets werefiltered for those included in the HG-U133A 2.0 gene chips.

A statistical analysis of the microarray data is performed to derive aplurality of hyperpigmentation gene expression signatures which maycomprise a statistically relevant number of the up-regulated anddown-regulated genes. In certain embodiments a hyperpigmentation geneexpression signature includes between 10 and 400 up-regulated and/orbetween 10 and 400 down-regulated genes. In more specific embodiments ahyperpigmentation gene expression signature includes the 50 moststatistically relevant up-regulated genes alone or in combination withthe 50 most statistically relevant down-regulated genes. Regulation isdetermined in comparison to gene expression in normal cells.

-   -   a. Filtering based on Absent/Margin/Present Calls. This filter        creates a list of potential genes for inclusion in the gene        expression signature. For example, a suitable filter may be that        at least 50% of the samples in one treatment group must have a        Present call for each probe set. Present calls are derived from        processing the raw GeneChip data and provide evidence that the        gene transcript complementary to a probe set that is actually        expressed in the biological sample. The probes that are absent        from all samples are likely to be just noisy measurements. This        step is important to filter out probe sets that do not        contribute meaningful data to the signature. For        hyperpigmentation gene expression signatures, the data was        filtered for probe sets with at least 50% Present calls provided        by the Affymetrix MAS 5 software.    -   b. Filtering According to a Statistical Measure. For example, a        suitable statistical measure may be p-values from a t-test,        ANOVA, correlation coefficient, or other model-based analysis.        As one example, p-values may be chosen as the statistical        measure and a cutoff value of p=0.05 may be chosen. Limiting the        signature list to genes that meet some reasonable cutoff for        statistical significance compared to an appropriate control is        important to allow selection of genes that are characteristic of        the biological state of interest. This is preferable to using a        fold change value, which does not take into account the noise        around the measurements. The t-statistic was used to select the        probe sets in the signatures because it is signed and provides        an indication of the directionality of the gene expression        changes (i.e. up- or down-regulated) as well as statistical        significance.    -   c. Sorting the Probe Sets. All the probe sets are sorted into        sets of up-regulated and down-regulated sets using the        statistical measure. For example, if a t-test was used to        compute p-values, the values (positive and negative) of the        t-statistic are used to sort the list since p-values are always        positive. The sorted t-statistics will place the sets with the        most significant p-values at the top and bottom of the list with        the non-significant ones near the middle.    -   d. Creation of the Gene expression signature. Using the filtered        and sorted list created, a suitable number of probe sets from        the top and bottom are selected to create a gene expression        signature that preferably has approximately the same number of        sets chosen from the top as chosen from the bottom. For example,        the gene expression signature created may have at least about        10, 50, 70, 100, 200, or 300 and/or less than about 800, 600,        400 or about 100 genes corresponding to a probe set on the chip.        The number of probe sets approximately corresponds to the number        of genes, but a single gene may be represented by more than one        probe set. It is understood that the phrase “number of genes” as        used herein, corresponds generally with the phrase “number of        probe sets.”

An exemplary Hyperpigmentation Condition Signature according to theinvention is provided, wherein the hyperpigmentation condition is SolarLentigines (Age Spots). Data is generated from an arm age spot genomicsstudy, full spot tissue vs. full normal tissue comparison. Probeselection method for up-regulated probes: 1. mean expression value ofspot tissue>200, 2. ratio of present call of spot tissue chips >=50%, 3.probe is significantly up regulated, p<0.05, 4. top 50, 100 and 200probes ranked by p values, are selected respectively. Three signaturesare generated using top up and down regulated probes cut at 50, 100 and200, respectively. C-Map hit evaluation: hits are selected based ontheir average weight from three signatures. Probe selection method fordown-regulated probes: 1. mean expression value of normal tissue>200, 2.ratio of present call of normal tissue chips >=50%, 3. probe issignificantly down regulated, p<0.05, 4. Top 50, 100 and 200 probesranked by p values, are selected respectively. The illustrativesignature is set forth as FIG. 2, Tables B and C.

Example 2

This Example provides support for the use of benchmark signatures in aC-map query to generate putative agents. The Example specificallyoutlines generation of a benchmark skin pigmentation-modifying geneexpression signature. As described herein, the benchmark skinpigmentation-modifying gene expression signature was generated usingmethods such as filtering as described in Example 1. More specifically,Hexamidine, N-acetyl glucosamine, Niacinamide, or SEPIWHITE (Sepiwhiteis the purported tradename of the agent known as undecylenoylphenylalanine) are applied as described below to tert-Keratinocyte cellsto generate the benchmark skin pigmentation-modifying gene expressionsignature.

A. Hexamidine (Hex).

A tert-Keratinocyte (tKC) cell line is used to conduct the genomicsstudy with an Affy U133A chip. (a) Probe selection method forup-regulated probes: 1. mean expression value of hex treated >200, 2.ratio of present calls of hex treated chips >=50%, 3. up regulated byhex, but down regulated by MSH (a skin darkening agent), 4. hex treatedp<0.05, top 100 probes ranked by p value; (b) Probe selection method fordown regulated probes, 1. mean expression value of DMSO control >200, 2.ratio of present calls of DMSO control chips >=50%, 3. down-regulated byhex, but up-regulated by MSH (a skin darkening agent), 4. hex treatedp<0.05, top 100 probes ranked by p value. The illustrative signaturesare set forth as FIGS. 10 and 11, Tables D and E, respectively.

B. N-acetyl-glucosamine (NAG).

tKC cell line, Affy U133A chip; Probe selection method for up-regulatedprobes: 1. mean expression value of NAG treated >200, 2. ratio ofpresent calls of NAG treated chips >=50%, 3. up regulated by NAG, butdown regulated by MSH (a skin darkening agent), 4. NAG treated p<0.05,39 probes are selected. Probe selection method for down-regulatedprobes: 1. mean expression value of DMSO control >200, 2. ratio ofpresent calls of DMSO control chips >=50%, 3. down regulated by NAG, butup regulated by MSH (a skin darkening agent), 4. NAG treated p<0.05, 43probes are selected. The illustrative signatures are set forth as FIGS.12 and 13, Tables F and G, respectively.

C. Niacinamide.

The Niainamide signature was generated by filtering analogous to thatused for hexamidine or NAG was used. The illustrative signatures are setforth as FIGS. 14 and 15, Tables H and I, respectively.

D. Sepiwhite.

The Sepiwhite signature was generated by filtering analogous to thatused for hexamidine or NAG was used. The illustrative signatures are setforth as FIGS. 16 and 17, Tables J and K, respectively.

Example 3

This Example illustrates use of C-map and the generation of a signatureas described in Example 2; however Example 3 outlines development of acomposite “skin tone” signature where Niacinaminde, Sepiwhite, NAG, andHexamidine are used together to generate a signature. This Exampleillustrates generation of an exemplary composite “skin tone” Signaturecomprised of four benchmark skin-lightening agents: Niacinamide,Sepiwhite, NAG, and Hexamidine. Chips Used for Signature Generation:DMSO control chips used for Signature generation: Conditions forSignature Generation: 1. a probe must have 10% present call amongControl chips or BenchMark chips, 2. The average signal for anup-regulated probe on the treated chip must be >200, 3. The averagesignal for a down-regulated probe on the control chip must be >200, 4. Aprobe must be up or down-regulated cross all benchmark chips. TableHeaders: Average signal of all control chips, AvgFC, AvgSignalTreated:Average signal of all treated chips, Average fold change,AvgSignalControl. The illustrative signature is set forth as FIG. 18,Table L.

This Example supports embodiments outlining how a composite signaturemay be generated by treating a cell sample with more than one agent. Asindicated earlier, a composite signature can be added in two ways: cellscan be treated with each agent separately, the signature can begenerated by comparing regulated genes from all agents (together),looking for genes regulated in the same direction by all agents;secondarily, agents can be mixed together prior to treatment of cells.In another embodiment, a composite benchmark signature may be generatedfor a skin-lightening agent, and another generated for a skin darkeningagent. The signature for the skin-lightening agent may be furthertweaked by eliminating any gene from the signature that also appears inthe signature of the skin-darkening agent, regulated in the samedirection, or vice versa. The inventors discovered that such compositesignatures are particularly useful for mining C-map for agents capableof modifying skin pigment in the desired direction.

Example 4

This Example illustrates use of C-map and the generation of a signature.More specifically, the signature was generated through application ofRetinoic acid to fibroblasts and keratinocytes. This Example illustratesa method for generating a benchmark skin tone agent signature accordingto the invention in each of two different cell types for comparison ofthe C-map hit evaluations, wherein the benchmark skin active agent isRetinoic acid (“RA”) and the cell types are (a) fibroblast, and (b)keratinocyte.

(a) Tert-keratinocytes (tKC) RA Signatures. Cells were treated with 1 uMtRA for 6 hr, tested in triplicate, with triplicate DMSO controls, andanalyzed on HG-U133A GeneChips; Signatures were generated like for BJfibroblasts, below; the signature KC_RA_200 consists of 100 mostsignificant up- and 100 most significant down-regulated probe sets; thesignature KC_RA_400 consists of 200 most significant up- and 200 mostsignificant down-regulated probe sets.(b) Fibroblast RA Signatures. The selected cell type is BJ fibroblast.Cells were treated with 1 uM tRA for 6 hr, tested in triplicate, withtriplicate DMSO controls, and analyzed on HG-U133A GeneChips. Presentcalls >0 for naïve, DMSO and RA (9 samples total). Mean signal >=200 forDMSO OR RA samples t-test p<0.05; Filtered for minimum fold change up ordown of 1.2; Used log fold change to establish directionality and sortedup and down lists by t-test p value. The signature BJ_RA_200 consists of100 most significant up- and 100 most significant down-regulated probesets; The signature BJ_RA_400 consists of 200 most significant up- and200 most significant down-regulated probe sets. The illustrativesignatures are set forth as FIGS. 19, 20, 21, AND 22, Tables M, N, O andP respectively.

Example 5

This example summarizes representative potential skin-lightening agentsand C-map query results for the benchmark skin active agentall-trans-retinoic acid according to the invention. C-map was queriedusing the Retinoic Acid/Keratinocyte 200 benchmark signature. Theaverage C-map scores for the top scoring known skin lightening agentsare tabled. Retinoic acid had the highest score of the materials testedbecause it was used to generate the signature. The data shown are forteleomerized human keratinocytes (tKC). Average CMap scores for someskin lightening agents with the Retinoic Acid Keratinocyte RA_200Signature are shown in FIG. 23, Table Q.

Example 6

This Example provides evidence of the advantages of using compositesignatures and illustrates clinical affirmation of the C-map model forpredicting efficacy of skin-lightening agents. This Example supportsembodiments related to composite signatures (as described at the end ofExample 3). An illustrative “Skin Tone” Signature developed from agenomics study using a composite skin-lightening benchmark agentsignature is derived from Niacinamide, Hexamidine, Sepiwhite, and NAG.The signature is used to query C-map and generate a list of potentialskin-lightening agents. A top hit, Chlorhexidine Diactate (CD) isentered into clinical testing for confirmation of efficacy. The controlfor clinical efficacy is a 5% Niacinamide+1% Sepiwhite formulation inthe control vehicle.

Primary endpoints are changes in color spot area fraction (imageanalysis) and melanin spot area fraction (NC2) from baseline. Secondaryendpoints are changes from baseline in L*a*b (color image analysis),mean melanin gray scale (NC2), and melanin evenness (NC2). Texture areafraction and pore area fraction are also evaluated to explore impact onother aspects relating to overall skin tone. Statistical significantsuperiority to the vehicle at one of these time points is a projectsuccess criteria. Statistically significant superiority to the highefficacy benchmark skin active agent to vehicle performance is theclinical success criteria.

Study Design: The experimental protocol included a 9-week (1-weekpreconditioning & 8-week treatment), randomized, double-blind, roundrobin, vehicle-controlled, split-face tone benefit study. The subjectpopulation included 330 Chinese females, 25-55 years old withhyperpigmented spots. 318 subjects completed the entire study.Pre-conditioning was achieved with application of Nature Science DeepPurify cleanser and study-specific moisturizer for a week. Olay CompleteSPF 15 UV Moisturizing Lotion is concomitantly used duringpre-conditioning and treatment. 0.5 g each test product per half-face(forehead to jaw line; ˜4 mg/cm²) is dosed 2× a day (morning/evening).Color SAF and treatment area, Melanin SAF, gray scale, evenness by NC2,and additional measurement of Fine Lines and Wrinkle and Texture are byREAL 3.01A. Data collection points include baseline, and ends of weeks4, 6 and 8. The tested hypothesis is that there is no difference inclinical endpoint versus the benchmark composition treatment. Study siteis Kuntai Clinical Center, Beijing, China and study time frame isFebruary 2010 (pre-conditioning) to April 2010.

0.05% Chlorhexidine Diacetate in SC-99 vehicle (7% glycerin) was a topconnectivity hit using the tone composite benchmark signature query setforth as Example 3 and is tested for clinical efficacy with respect tofour different tone criteria against a known high efficacy benchmarkcomposition of 5% Niacinamide and 1% Sepiwhite in SC-99 vehicle.

Results:

According to the spot area fraction color test: 0.05% CD showedsignificantly fewer spots when compared to vehicle at week 8. In thespot area fraction NC2 test, CD showed a significantly reduced fractionwhen compared to the vehicle at both weeks 6 and 8. In the NC2 Melaninevenness test, CD demonstrated superiority to the vehicle at weeks 6 and8, and with respect to Basal skin tone, CD demonstrated superiority atweek 8. Surprisingly, CD also demonstrated superior efficacy in the Porearea and Texture area fractions when compared to the vehicle at week 8,suggesting that it is a good candidate for overall skin tone and textureenhancement.

Example 7

This Example provides support for the unexpected effectiveness ofcomposite signature use with the C-map technology, with this Exampleillustrating a comparison of expression signature efficacy in predictinginhibitors; specifically, from 33 various chemicals identified asmelanogenesis inhibitors in the mouse B16 melanoma cell assay (FIG. 24,Table R). The composite signature was unexpectedly more effective atidentifying inhibitors than any of the individual signatures (such asfor Niacinamide, NAG, Hexamidine, or Sepiwhite). For this analysis C-maphits were defined as materials occurring in the top 200 instances (fromthe same pool of 2266 instances) with a score 0.30. Result Summary ofcorrectly predicted melanogenesis inhibitors: Composite signature: 20,Niacinamide: 1, NAG: 1, Hexamidine: 2, and Sepiwhite: 9. The C-mapscores shown in Table R are average scores across the instances of thechemicals. A maximum positive C-map score is 2.0 indicating perfectpositive connectivity. The individual materials do not show perfectlyhigh scores linking to themselves because of replicate variability,which is more evident for materials with relatively weak effects on geneexpression. Surprisingly, for this set of materials the compositesignature in 3 of 4 cases gave better scores with the benchmarkmaterials than the individual benchmark signatures. The process ofgenerating the benchmark signature may select for the most consistentlyregulated probe sets, which may account for this result.

As indicated in Example 3, this Example (7) supports embodimentsoutlining how a composite signature may be generated by treating a cellsample with more than one agent. As indicated earlier, a compositesignature can be added in two ways: cells can be treated with each agentseparately, the signature can be generated by comparing regulated genesfrom all agents (together), looking for genes regulated in the samedirection by all agents; secondarily, agents can be mixed together priorto treatment of cells. In another embodiment, a composite benchmarksignature may be generated for a skin-lightening agent, and anothergenerated for a skin darkening agent. The signature for theskin-lightening agent may be further tweaked by eliminating any genefrom the signature that also appears in the signature of theskin-darkening agent, regulated in the same direction, or vice versa.The inventors discovered that such composite signatures are particularlyuseful for mining C-map for agents capable of modifying skin pigment inthe desired direction.

Example 8

This Example provides support to illustrate that it is believed thatkeratinocyte cells, rather than melanocyte or melanoma cells, haveexhibited a more robust transcriptional profile when treated withskin-lightening agents. Keratinocytes have been preliminarily shown tobe easier to grow than melanocytes and have increased responsivenesssuch that keratinocytes may be able to be used to detect activechemicals over a wider range of concentrations than testing withmelanocytes. More specifically, in this Example, six skin tone benchmarkmaterials were applied to each of three cell types (tert-keratinocytes,melanocyes, and melanoma cells), and with four of six tested materialstert-keratinocytes showed the greatest response (as indicated in FIG.25, Table S which shows that with four of the six tested materials, thenumber of probe sets with significant P-values compared to DMSO controlswas greatest for tert-keratinocytes). As can be seen in Table S, The sixtested skin tone benchmark materials included: Haxamidine diisothionate,Myo-inositol, N-acetyl-glucosamine, NDP-MSH, Niacinamide, and Sepiwhite.For completeness, details of the exact types of melanocytes and melanomacells, as well as the cell culturing conditions and result analysis areprovided herein below.

HEMn primary neonatal medium pigment melanocytes were obtained fromInvitrogen, Carlsbad, Calif. and were cultured in Medium 254 fromInvitrogen. HBL melanoma cells were obtained from the Laboratory ofOncology and Experimental Surgery, Institut Bordet, Universite' Libre deBruxelles, Belgium and were cultured in F-10 Nutrient Mixture (Ham) fromInvitrogen supplemented with 10% fetal bovine serum (HyClone, Logan,Utah). Human telomerized keratinocytes (tert-keratinocytes) wereobtained from the University of Texas, Southwestern Medical Center,Dallas, Tex. and were grown in EpiLife® media with 1× Human KeratinocyteGrowth Supplement (Invitrogen). All cells were incubated at 37° C. in ahumidified incubator with 5% CO2.

Cells were seeded into 6-well plates a 24 hours before chemicalexposure, and the skin tone benchmark chemicals listed in the tablebelow were added to culture medium dissolved in DMSO. The finalconcentration of DMSO was 0.1%, and cells treated just with DMSO servedas controls. After 6 hours of chemical exposure cells were then lysedwith 350 ul/well of RLT buffer containing β-mercaptoethanol (Qiagen,Valencia, Calif.), transferred to a 96-well plate, and stored at −20° C.RNA from cell culture batches was isolated from the RLT buffer usingAgencourt® RNAdvance Tissue-Bind magnetic beads (Beckman-Coulter, BreaCalif. 92821) according to manufacturer's instructions. 1 ug of totalRNA per sample was labeled using Ambion Message Amp™ II Biotin Enhancedkit (Life Technologies, Grand Island, N.Y. 14072) according tomanufacturer's instructions. The resultant biotin labeled and fragmentedcRNA was hybridized to an Affymetrix HG-U133A 2.0 GeneChip®, which wasthen washed, stained and scanned using the protocol provided byAffymetrix.

Regarding the results and analysis of the testing: Two sample t-testswere performed on each treatment to compare with the DMSO control. Thenumber of probe sets with significant p-values (<0.05) are summarized inthe table below. Each GeneChip® contains 22215 probes sets. Using asignificance level of 0.05, 1111 probe sets (95% confidence interval of1047 to 1174) are expected to be significant by chance alone. Thisestimate is somewhat conservative since there may be multiple probe setsfor the same gene.

In summary, the tert-keratinocytes were generally the most responsivecells to the skin tone benchmark materials. There were moresignificantly regulated probe sets for 4/6 skin tone benchmark materialsin the tert-keratinocytes compared to either HeMnMP melanocytes or HBLmelanoma cells. The tert-keratinocytes have an additional advantage overthe second most responsive cells, HeMnMP melanocytes, in that they growsubstantially faster and are more practical cell line for routinescreening.

Every document cited herein is hereby incorporated herein by referencein its entirety unless expressly excluded or otherwise limited. Thecitation of any document is not an admission that it is prior art withrespect to any invention disclosed or claimed herein or that it alone,or in any combination with any other reference or references, teaches,suggests or discloses any such invention. Further, to the extent anymeaning or definition of a term in this document conflicts with anymeaning or definition of the same term in a document incorporated byreference, the meaning or definition assigned to that term in thisdocument shall govern.

The values disclosed herein are not to be understood as being strictlylimited to the exact numerical values recited. Instead, unless otherwisespecified, each such value is intended to mean both the recited valueand a functionally equivalent range surrounding that value.

The present invention should not be considered limited to the specificexamples described herein, but rather should be understood to cover allaspects of the invention. Various modifications, equivalent processes,as well as numerous structures and devices to which the presentinvention may be applicable will be readily apparent to those of skillin the art. Those skilled in the art will understand that variouschanges may be made without departing from the scope of the invention,which is not to be considered limited to what is described in thespecification.

What is claimed is:
 1. A method of constructing a database for use inidentifying connections between perturbagens and genes associated withskin tone, comprising: (a) providing a gene expression profile for acontrol human cell, wherein the control cell is from a human cell lineselected from the group consisting of keratinocyte, fibroblast,melanocyte and melanoma cell lines; (b) generating a gene expressionprofile for a human cell exposed to at least one perturbagen, whereinthe perturbagen comprises a skin active agent that modifies skinpigmentation, and wherein the cell is from the same cell line as thecontrol cell; (c) identifying genes differentially expressed in responseto the at least one perturbagen by comparing the gene expressionprofiles of (a) and (b); (d) creating an ordered list comprisingidentifiers representing the differentially expressed genes, wherein theidentifiers are ordered according to the differential expression of thegenes; (e) storing the ordered list as an instance on a non-transitorycomputer readable medium, wherein the instance includes metadataidentifying the cell line from the selection in (a); and (f)constructing a database of stored instances by repeating (a) through (e)for between about 50 and about 50,000 instances, wherein the at leastone perturbagen of step (b) is different or quantitatively for eachinstance, and steps c, d, e, and f are performed using a programmablecomputer.
 2. The method of claim 1, wherein generating a gene expressionprofile for a human cell exposed to at least one perturbagen comprisesextracting a biological sample from the exposed cell and subjecting thebiological sample to microarray analysis.
 3. The method of claim 1,wherein the skin active agent is selected from an anti-inflammatoryagent that suppresses skin pigmentation, an environmental stimuli thatinduces or suppresses skin pigmentation, and an agent that antagonizesendocrine induction of pigmentation.
 4. The method of claim 1, whereinthe ordered list of each instance is arranged so that an identifierassociated with each gene that is not differentially expressed ispositioned between the identifier associated with the most up-regulatedgene and the identifier associated with the most down-regulated gene. 5.A method of implementing the database of claim 1 to identify at leastone putative skin active agent for treating a skin hyperpigmentationcondition, the method comprising: (a) querying the database with a geneexpression signature, wherein (i) querying comprises comparing the geneexpression signature to each stored instance and assigning aconnectivity score to each comparison, wherein the connectivity score isa combination of an up-score and a down-score, the up-score representingthe correlation between up-regulated genes of the gene signature and thestored instance and the down-score representing the correlation betweenthe down-regulated genes of the gene signature and the stored instance,(ii) the gene expression signature represents genes differentiallyexpressed in cells derived from skin affected with a skinhyperpigmentation condition or genes differentially expressed in cellstreated with at least one benchmark skin active agent having knownefficacy in treating a skin hyperpigmentation condition, and (iii) thegene expression signature is derived from the same cell line as theinstances; and (b) identifying a perturbagen associated with theinstance as a putative skin active agent for treating thehyperpigmentation condition when the connectivity score is positive, andidentifying the perturbagen associated with the instance as a putativeskin active agent for darkening skin when the connectivity score isnegative.
 6. The method of claim 5, wherein the hyperpigmentationcondition results from one or more of a post-inflammatory response, age,exposure to UVB radiation, endocrine induction of pigmentation, and ahost skin predisposition.
 7. The method of claim 5, wherein the geneexpression signature is constructed by a method comprising (i)identifying genes having up-regulated expression in thehyperpigmentation condition when compared to a control; (ii) identifyinggenes having down-regulated expression in the hyperpigmentationcondition when compared to a control; (iii) creating one or more geneexpression signature lists comprising identifiers corresponding to aplurality of the genes identified in (i) and (ii); and storing the oneor more gene expression signature lists on the at least onenon-transitory computer readable medium.
 8. The method of claim 7,wherein the hyperpigmentation condition is solar lentigines and thenumber of genes having up-regulated expression in the hyperpigmentationcondition is between about 10 and about 500, and the number of genesdown-regulated in the hyperpigmentation condition is between about 10and about
 500. 9. The method of claim 8, wherein the identifiers forabout 80% to about 100% of the up-regulated genes are set forth as inTable B and wherein identifiers for about 80% to about 100% of thedown-regulated genes are set forth in Table C.
 10. The method of claim7, wherein the one or more gene expression signature lists comprises afirst list representing a plurality of the up-regulated genes identifiedin (i) of claim 7 and a second list representing a plurality ofdown-regulated genes identified in (ii) of claim
 7. 11. The method ofclaim 7, wherein at least one skin sample is taken from a human subjectexhibiting the hyperpigmentation condition, a biological sample isextracted from the skin sample, and a gene expression profile of the atleast one skin sample is generated prior to at least one of (i) of claim7 and (ii) of the claim
 7. 12. The method of claim 5, wherein the geneexpression signature is a benchmark skin-lightening agent geneexpression signature and is constructed by a method comprising (i)identifying genes up-regulated in a skin cell treated with the benchmarkskin-lightening agent when compared to a control; (ii) identifying genesdown-regulated in a skin cell treated with the benchmark skin-lighteningagent when compared to a control; (iii) creating one or more geneexpression signature lists comprising identifiers corresponding to aplurality of genes identified in (i) and (ii); and storing the one ormore gene expression signature lists on the at least one non-transitorycomputer readable medium.